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.

Case report: a genomics-guided reclassification of a blood culture isolate misassigned by MALDI-TOF as Yersinia pestis.

In this report, we describe a case where Gram-negative rods were isolated from a blood culture which subsequently presented a discordant Yersinia species result by MALDI-TOF. Rapid sequencing provided high-resolution identification of the isolate as Yersinia pseudotuberculosis , which was subsequently confirmed by biochemical tests.

COVID-19 Infection With the Omicron SARS-CoV-2 Variant in a Cohort of Kidney and Kidney Pancreas Transplant Recipients: Clinical Features, Risk Factors, and Outcomes.

BACKGROUND: Since November 2021, a new variant of concern (VOC), the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineage B.1.1.529 (Omicron) has emerged as the dominant coronavirus disease 2019 (COVID-19) infection worldwide. We describe the clinical presentation, risk factors, and outcomes in a cohort of kidney and kidney pancreas transplant recipients with COVID-19 caused by Omicron infection. METHODS: We included all kidney and kidney pancreas transplant recipients diagnosed with SARS-CoV-2 Omicron infections between December 26, 2021, and January 14, 2022, in a single transplant center in Australia. Identification of the VOC Omicron was confirmed using phylogenetic analysis of SARS-CoV-2 sequences. RESULTS: Forty-one patients with kidney (6 living and 33 deceased) and kidney pancreas transplants were diagnosed with the VOC Omicron (lineage B.1.1.529/BA.1) infection during the study period. The mean age (SD) at the time of diagnosis was 52 (11.1) y; 40 (out of 41) (98%) had received at least 2 doses of COVID-19 vaccine. Cough was the most frequent symptom (80.5%), followed by myalgia (70.7%), sore throat (63.4%), and fever (58.5%). After a follow-up time of 30 d, 1 (2.4%) patient died, 2 (4.9%) experienced multiorgan failure, and 5 (12.2%) had respiratory failure; 11 (26.8%) patients developed other superimposed infections. Compared with recipients who did not receive sotrovimab antibody therapy, the odds ratio (95% confidence interval) for hospitalization among patients who received sotrovimab was 0.05 (0.005-0.4). CONCLUSIONS: Despite double or triple dose vaccination, VOC Omicron infections in kidney and kidney pancreas transplant recipients are not necessarily mild. Hospitalization rates remained high (around 56%), and sotrovimab use may prevent hospitalization.

Co-infection with SARS-CoV-2 Omicron and Delta variants revealed by genomic surveillance.

Co-infections with different variants of SARS-CoV-2 are a key precursor to recombination events that are likely to drive SARS-CoV-2 evolution. Rapid identification of such co-infections is required to determine their frequency in the community, particularly in populations at-risk of severe COVID-19, which have already been identified as incubators for punctuated evolutionary events. However, limited data and tools are currently available to detect and characterise the SARS-CoV-2 co-infections associated with recognised variants of concern. Here we describe co-infection with the SARS-CoV-2 variants of concern Omicron and Delta in two epidemiologically unrelated adult patients with chronic kidney disease requiring maintenance haemodialysis. Both variants were co-circulating in the community at the time of detection. Genomic surveillance based on amplicon- and probe-based sequencing using short- and long-read technologies identified and quantified subpopulations of Delta and Omicron viruses in respiratory samples. These findings highlight the importance of integrated genomic surveillance in vulnerable populations and provide diagnostic pathways to recognise SARS-CoV-2 co-infection using genomic data.

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.

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.

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.

PCR-based tests for the early diagnosis of sepsis. Where do we stand?

PURPOSE OF REVIEW: Bloodstream infections are a major cause of hospital and ICU admission with high morbidity and mortality; however, early and targeted antimicrobial therapy reduces mortality in high-risk patients. This article focuses on the diagnosis of bloodstream infections by PCR-based approaches at an early stage to enable prompt treatment and prevent organ dysfunction. RECENT FINDINGS: PCR systems offering highly multiplexed targeting of bacterial and/or fungal pathogens (in whole blood) offer the best opportunity for clinical impact, as informed decisions can be made within 4-8 h of the blood draw. Although more rapid, these systems are typically associated with lower sensitivity and specificity than postculture detection methods which rely on microbial growth. Additionally, unlike postculture methods, detection directly from blood is not prone to misleading results because of concurrent (or previous) therapy, which limit clinical relevance. SUMMARY: Rapid and accurate identification of the cause of sepsis is essential in improving patient outcomes. Early identification of these pathogens by nucleic acid detection assays directly from blood samples remains key to achieving this, particularly if taken at the time of presentation. Selection of the most suitable PCR system is typically influenced by local epidemiology and by the resources of the testing laboratory.

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.

Pre- and post-weaning scours in southeastern Australia: A survey of 22 commercial pig herds and characterisation of Escherichia coli isolates.

Diarrhoeal diseases in piglets caused by Escherichia coli are responsible for substantial losses each year in the Australian pig industry. A cross-sectional survey was conducted (September 2013-May 2014) across 22 commercial pig herds located in southeastern Australia: NSW (n = 9); VIC (n = 10); and SA (n = 3), to estimate the prevalence of E. coli associated diarrhoea in pre- and post-weaned piglets and to identify key risk factors associated with E. coli disease. A questionnaire on management and husbandry practices was included. Faecal samples (n = 50 from each herd) were tested for the presence of ß-haemolytic E. coli. Species level identification was confirmed by matrix-assisted laser desorption / ionisation time-of-flight mass spectrometry (MALDI-TOF/MS). ETEC virulence and enterotoxin genes (F4, F5, F6, F18, F41, STa, STb and LT) were screened for by multiplex PCR. This study assessed 60 potential risk factors for E. coli disease in post-weaned piglets, with 2 key factors-recent disease events and the presence of bedding, statistically associated with the presence of post-weaning scours. The prevalence of diarrhea in pre-weaned pens was 17% (16/93), compared with 24% (24/102) in post-weaned pens. The most prevalent ß-haemolytic ETEC genes were F18 (32%) and STb (32%) but isolates were more likely to contain F4:STb (11 of 22 herds, 23%), than F18:STb (5 of 22 herds, 6%). These findings indicate that recent disease events that have occurred within the last 12 months, and by the use of bedding or not maintaining fresh bedding can have significant impacts on piglet diarrhoea.

Quantitative multiplexed-tandem PCR for direct detection of bacteraemia in critically ill patients.

Culture remains the gold standard for diagnosis of blood stream infections (BSI), but its clinical utility is limited by slow turnaround times. Here we describe a method for rapid quantitative detection of bacterial DNA directly extracted from whole blood using a multiplexed tandem real-time PCR (MT-PCR) assay targeting Staphylococcus, Streptococcus, Pseudomonas, Enterococcus and Enterobacteriaceae 16S rDNA genes. Results were available less than 3.5 hours after blood collection with all five bacterial targets having limits of detection between 101 and 103 CFU/mL. A small-scale clinical evaluation of the assay using blood samples collected from 15 patients admitted to the Intensive Care Unit at our institution demonstrated 93.3% (14/15) concordance between MT-PCR and blood culture when detection of persistent bacterial DNAemia by MT- PCR was considered a true result. Further evaluation with clinical samples is needed; however, this method has potential as an effective rule-in diagnostic tool for bacteraemic sepsis and septic shock.

Predictability of Phenotype in Relation to Common ß-Lactam Resistance Mechanisms in Escherichia coli and Klebsiella pneumoniae.

The minimal concentration of antibiotic required to inhibit the growth of different isolates of a given species with no acquired resistance mechanisms has a normal distribution. We have previously shown that the presence or absence of transmissible antibiotic resistance genes has excellent predictive power for phenotype. In this study, we analyzed the distribution of six ß-lactam antibiotic susceptibility phenotypes associated with commonly acquired resistance genes in Enterobacteriaceae in Sydney, Australia. Escherichia coli (n = 200) and Klebsiella pneumoniae (n = 178) clinical isolates, with relevant transmissible resistance genes (blaTEM, n = 33; plasmid AmpC, n = 69; extended-spectrum ß-lactamase [ESBL], n = 116; and carbapenemase, n = 100), were characterized. A group of 60 isolates with no phenotypic resistance to any antibiotics tested and carrying none of the important ß-lactamase genes served as comparators. The MICs for all drug-bacterium combinations had a normal distribution, varying only in the presence of additional genes relevant to the phenotype or, for ertapenem resistance in K. pneumoniae, with a loss or change in the outer membrane porin protein OmpK36. We demonstrated mutations in ompK36 or absence of OmpK36 in all isolates in which reduced susceptibility to ertapenem (MIC, >1 mg/liter) was evident. Ertapenem nonsusceptibility in K. pneumoniae was most common in the context of an OmpK36 variant with an ESBL or AmpC gene. Surveillance strategies to define appropriate antimicrobial therapies should include genotype-phenotype relationships for all major transmissible resistance genes and the characterization of mutations in relevant porins in organisms, like K. pneumoniae.

MALDI-TOF Mass Spectrometry for Multilocus Sequence Typing of Escherichia coli Reveals Diversity among Isolates Carrying blaCMY₋2-Like Genes.

Effective surveillance and management of pathogenic Escherichia coli relies on robust and reproducible typing methods such as multilocus sequence typing (MLST). Typing of E. coli by MLST enables tracking of pathogenic clones that are known to carry virulence factors or spread resistance, such as the globally-prevalent ST131 lineage. Standard MLST for E. coli requires sequencing of seven alleles, or a whole genome, and can take several days. Here, we have developed and validated a nucleic-acid-based MALDI-TOF mass spectrometry (MS) method for MLST as a rapid alternative to sequencing that requires minimal operator expertise. Identification of alleles was 99.6% concordant with sequencing. We employed MLST by MALDI-TOF MS to investigate diversity among 62 E. coli isolates from Sydney, Australia, carrying a blaCMY-2-like gene on an IncI1 plasmid to determine whether any dominant clonal lineages are associated with the spread of this globally-disseminated resistance gene. Thirty-four known sequence types were identified, including lineages associated with human disease, animal and environmental sources. This suggests that the dissemination of blaCMY-2-like-genes is more complex than the simple spread of successful pathogenic clones. E. coli MLST by MALDI-TOF MS, employed here for the first time, can be utilised as an automated tool for large-scale population analyses or for targeted screening for known high-risk clones in a diagnostic setting.

Relative Strengths of Promoters Provided by Common Mobile Genetic Elements Associated with Resistance Gene Expression in Gram-Negative Bacteria.

Comparison of green fluorescent protein expression from outward-facing promoters (POUT) of ISAba1, ISEcp1, and ISAba125 revealed approximate equivalence in strength, intermediate between PCS (strong) and PCWTGN-10 (weak) class 1 integron promoter variants, >30-fold stronger than POUT of ISCR1, and >5 times stronger than Ptac. Consistent with its usual role, PCWTGN-10 produces more mRNA from a "downstream" gfp gene transcriptionally linked to a "usual" PCWTGN-10-associated gene cassette than does POUT of ISAba1.

Distribution of acquired AmpC ß-lactamase genes in Sydney, Australia.

Investigation of plasmid-borne AmpC ß-lactamase genes in Escherichia coli and Klebsiella spp. revealed blaCMY-2-like genes predominantly in E. coli and blaDHA genes equally distributed between both species. This distribution remained stable over time, but blaACT/MIR-like genes, initially common in Klebsiella spp., were not identified in more recent isolates.

Different IncI1 plasmids from Escherichia coli carry ISEcp1-blaCTX-M-15 associated with different Tn2-derived elements.

The bla(CTX-M-15) gene, encoding the globally dominant CTX-M-15 extended-spectrum ß-lactamase, has generally been found in a 2.971-kb ISEcp1-bla(CTX-M-15)-orf477Δ transposition unit, with ISEcp1 providing a promoter. In available IncF plasmid sequences from Escherichia coli, this transposition unit interrupts a truncated copy of transposon Tn2 that lies within larger multiresistance regions. In E. coli, bla(CTX-M-15) is also commonly associated with IncI1 plasmids and here three such plasmids from E. coli clinical isolates from western Sydney 2006-2007 have been sequenced. The plasmid backbones are organised similarly to those of other IncI1 plasmids, but have insertions and/or deletions and sequence differences. Each plasmid also has a different insertion carrying bla(CTX-M-15). pJIE113 (IncI1 sequence type ST31) is almost identical to plasmids isolated from the 2011 E. coli O104:H4 outbreak in Europe, where the typical bla(CTX-M-15) transposition unit interrupts a complete Tn2 inserted directly in the plasmid backbone. In the novel plasmid pJIE139 (ST88), ISEcp1-blaC(TX-M-15)-orf477Δ lies within a Tn2/3 hybrid transposon. Homologous recombination could explain movement of ISEcp1-bla(CTX-M-15)-orf477Δ between copies of Tn2 on IncF and IncI1 plasmids and generation of the Tn2/3 hybrid. pJIE174 (ST37) is almost identical to pESBL-12 from the Netherlands and in these plasmids bla(CTX-M-15) is flanked by two copies of IS26 that truncate the transposition unit within a larger region bounded by the ends of Tn2. bla(CTX-M-15) and the associated ISEcp1-derived promoter may be able to move from this structure by the actions of IS26, independently of both ISEcp1 and Tn2.

Rapid identification of pathogens using molecular techniques.

Real-time PCR is the traditional face of nucleic acid detection in the diagnostic microbiology laboratory and is now generally regarded as robust enough to be widely adopted. Methods based on nucleic acid detection of this type are bringing increased accuracy to diagnosis in areas where culture is difficult and/or expensive, and these methods are often effective partners to other rapid molecular diagnostic tools such as matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS). This change in practice has particularly affected the recognition of viruses and fastidious or antibiotic-exposed bacteria, but has been also shown to be effective in the recognition of troublesome or specialised phenotypes such as antiviral resistance and transmissible antibiotic resistance in the Enterobacteriaceae. Quantitation and high-intensity sequencing (of multiple whole genomes) has brought new opportunities as well as new challenges to the microbiology community. Diagnostic microbiologists currently training might be expected to deal less with the culture-based techniques of the last half-century than with the high-volume data and complex analyses of the next.

Emergence of blaKPC carbapenemase genes in Australia.

blaKPC genes encoding resistance to carbapenems are increasingly widely reported and are now endemic in parts of several countries, but only one Klebsiella pneumoniae isolate carrying blaKPC-2 had previously been reported in Australia, in 2010. Here we characterised this isolate, six additional K. pneumoniae and one Escherichia coli carrying blaKPC and another K. pneumoniae lacking blaKPC, all isolated in Australia in 2012. Seven K. pneumoniae belonged to clonal complex (CC) 292, associated with blaKPC in several countries. Five with blaKPC-2 plus the isolate lacking a blaKPC gene were sequence type 258 (ST258) and the seventh was the closely related ST512 with blaKPC-3. The eighth K. pneumoniae isolate, novel ST1048, and the E. coli (ST131) also carried blaKPC-2. blaKPC genes were associated with the most common Tn4401a variant, which gives the highest levels of expression, in all isolates. The ST258 isolates appeared to share a similar set of plasmids, with IncFIIK, IncX3 and ColE-type plasmids identified in most isolates. All K. pneumoniae isolates had a characteristic insertion in the ompK35 gene resulting in a frameshift and early termination, but only the ST512 isolate had a GlyAsp insertion in loop 3 of OmpK36 that may contribute to increased resistance. The clinical epidemiology of blaKPC emergence in Australia thus appears to reflect the global dominance of K. pneumoniae CC292 (and perhaps E. coli ST131). Some, but not all, patients carrying these isolates had previously been hospitalised outside Australia, suggesting multiple discrete importation events of closely related strains, as well as undetected nosocomial spread.