Multidrug-resistant OXA-48/CTX-M-15 Klebsiella pneumoniae cluster in a COVID-19 intensive care unit: salient lessons for infection prevention and control during the COVID-19 pandemic.

BACKGROUND: Wards caring for COVID-19 patients, including intensive care units (ICUs), have an important focus on preventing transmission of SARS-CoV-2 to other patients and healthcare workers. AIM: To describe an outbreak of carbapenemase-producing Enterobacterales (CPE) in a COVID-19 ICU and to discuss key infection control measures enabling prompt termination of the cluster. METHODS: CPE were isolated from clinical specimens and screening swabs from intensive care patients with COVID-19 disease and from environmental screening. Whole-genome sequencing analysis was instrumental in informing phylogenetic relationships. FINDINGS: Seven clinical isolates and one environmental carbapenemase-producing Klebsiella pneumoniae isolate - all carrying OXA-48, CTX-M-15 and outer membrane porin mutations in ompK35/ompK36 - were identified with ≤1 single nucleotide polymorphism difference, indicative of clonality. A bundle of infection control interventions including careful adherence with contact precautions and hand hygiene, twice weekly screening for multidrug-resistant organisms, strict antimicrobial stewardship, and enhanced cleaning protocols promptly terminated the outbreak. CONCLUSION: Prolonged use of personal protective equipment is common with donning and doffing stations at the ward entrance, leaving healthcare workers prone to reduced hand hygiene practices between patients. Minimizing transmission of pathogens other than SARS-CoV-2 by careful adherence to normal contact precautions including hand hygiene, even during high patient contact manoeuvres, is critical to prevent outbreaks of multidrug-resistant organisms. Appropriate antimicrobial stewardship and screening for multidrug-resistant organisms must also be maintained throughout surge periods to prevent medium-term escalation in antimicrobial resistance rates. Whole-genome sequencing is highly informative for multidrug-resistant Enterobacterales surveillance strategies.

Association Between Minimum Inhibitory Concentration, Beta-lactamase Genes and Mortality for Patients Treated With Piperacillin/Tazobactam or Meropenem From the MERINO Study.

INTRODUCTION: This study aims to assess the association of piperacillin/tazobactam and meropenem minimum inhibitory concentration (MIC) and beta-lactam resistance genes with mortality in the MERINO trial. METHODS: Blood culture isolates from enrolled patients were tested by broth microdilution and whole genome sequencing at a central laboratory. Multivariate logistic regression was performed to account for confounders. Absolute risk increase for 30-day mortality between treatment groups was calculated for the primary analysis (PA) and the microbiologic assessable (MA) populations. RESULTS: In total, 320 isolates from 379 enrolled patients were available with susceptibility to piperacillin/tazobactam 94% and meropenem 100%. The piperacillin/tazobactam nonsusceptible breakpoint (MIC >16 mg/L) best predicted 30-day mortality after accounting for confounders (odds ratio 14.9, 95% confidence interval [CI] 2.8-87.2). The absolute risk increase for 30-day mortality for patients treated with piperacillin/tazobactam compared with meropenem was 9% (95% CI 3%-15%) and 8% (95% CI 2%-15%) for the original PA population and the post hoc MA populations, which reduced to 5% (95% CI -1% to 10%) after excluding strains with piperacillin/tazobactam MIC values >16 mg/L. Isolates coharboring extended spectrum ß-lactamase (ESBL) and OXA-1 genes were associated with elevated piperacillin/tazobactam MICs and the highest risk increase in 30-day mortality of 14% (95% CI 2%-28%). CONCLUSIONS: After excluding nonsusceptible strains, the 30-day mortality difference from the MERINO trial was less pronounced for piperacillin/tazobactam. Poor reliability in susceptibility testing performance for piperacillin/tazobactam and the high prevalence of OXA coharboring ESBLs suggests that meropenem remains the preferred choice for definitive treatment of ceftriaxone nonsusceptible Escherichia coli and Klebsiella.

Whole-genome analysis of extraintestinal Escherichia coli sequence type 73 from a single hospital over a 2 year period identified different circulating clonal groups.

Sequence type (ST)73 has emerged as one of the most frequently isolated extraintestinal pathogenic Escherichia coli. To examine the localized diversity of ST73 clonal groups, including their mobile genetic element profile, we sequenced the genomes of 16 multiple-drug resistant ST73 isolates from patients with urinary tract infection from a single hospital in Sydney, Australia, between 2009 and 2011. Genome sequences were used to generate a SNP-based phylogenetic tree to determine the relationship of these isolates in a global context with ST73 sequences (n=210) from public databases. There was no evidence of a dominant outbreak strain of ST73 in patients from this hospital, rather we identified at least eight separate groups, several of which reoccurred, over a 2 year period. The inferred phylogeny of all ST73 strains (n=226) including the ST73 clone D i2 reference genome shows high bootstrap support and clusters into four major groups that correlate with serotype. The Sydney ST73 strains carry a wide variety of virulence-associated genes, but the presence of iss, pic and several iron-acquisition operons was notable.

The role of whole genome sequencing in antimicrobial susceptibility testing of bacteria: report from the EUCAST Subcommittee.

Whole genome sequencing (WGS) offers the potential to predict antimicrobial susceptibility from a single assay. The European Committee on Antimicrobial Susceptibility Testing established a subcommittee to review the current development status of WGS for bacterial antimicrobial susceptibility testing (AST). The published evidence for using WGS as a tool to infer antimicrobial susceptibility accurately is currently either poor or non-existent and the evidence / knowledge base requires significant expansion. The primary comparators for assessing genotypic-phenotypic concordance from WGS data should be changed to epidemiological cut-off values in order to improve differentiation of wild-type from non-wild-type isolates (harbouring an acquired resistance). Clinical breakpoints should be a secondary comparator. This assessment will reveal whether genetic predictions could also be used to guide clinical decision making. Internationally agreed principles and quality control (QC) metrics will facilitate early harmonization of analytical approaches and interpretive criteria for WGS-based predictive AST. Only data sets that pass agreed QC metrics should be used in AST predictions. Minimum performance standards should exist and comparative accuracies across different WGS laboratories and processes should be measured. To facilitate comparisons, a single public database of all known resistance loci should be established, regularly updated and strictly curated using minimum standards for the inclusion of resistance loci. For most bacterial species the major limitations to widespread adoption for WGS-based AST in clinical laboratories remain the current high-cost and limited speed of inferring antimicrobial susceptibility from WGS data as well as the dependency on previous culture because analysis directly on specimens remains challenging. For most bacterial species there is currently insufficient evidence to support the use of WGS-inferred AST to guide clinical decision making. WGS-AST should be a funding priority if it is to become a rival to phenotypic AST. This report will be updated as the available evidence increases.

In vitro volatile organic compound profiling using GC×GC-TOFMS to differentiate bacteria associated with lung infections: a proof-of-concept study.

Chronic pulmonary infections are the principal cause of morbidity and mortality in individuals with cystic fibrosis (CF). Due to the polymicrobial nature of these infections, the identification of the particular bacterial species responsible is an essential step in diagnosis and treatment. Current diagnostic procedures are time-consuming, and can also be expensive, invasive and unpleasant in the absence of spontaneously expectorated sputum. The development of a rapid, non-invasive methodology capable of diagnosing and monitoring early bacterial infection is desired. Future visions of real-time, in situ diagnosis via exhaled breath testing rely on the differentiation of bacteria based on their volatile metabolites. The objective of this proof-of-concept study was to investigate whether a range of CF-associated bacterial species (i.e. Pseudomonas aeruginosa, Burkholderia cenocepacia, Haemophilus influenzae, Stenotrophomonas maltophilia, Streptococcus pneumoniae and Streptococcus milleri) could be differentiated based on their in vitro volatile metabolomic profiles. Headspace samples were collected using solid phase microextraction (SPME), analyzed using comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (GC×GC-TOFMS) and evaluated using principal component analysis (PCA) in order to assess the multivariate structure of the data. Although it was not possible to effectively differentiate all six bacteria using this method, the results revealed that the presence of a particular pattern of VOCs (rather than a single VOC biomarker) is necessary for bacterial species identification. The particular pattern of VOCs was found to be dependent upon the bacterial growth phase (e.g. logarithmic versus stationary) and sample storage conditions (e.g. short-term versus long-term storage at -18 °C). Future studies of CF-associated bacteria and exhaled breath condensate will benefit from the approaches presented in this study and further facilitate the production of diagnostic tools for the early detection of bacterial lung infections.

Delusional infestation: an Australian multicentre study of 23 consecutive cases.

Delusional infestation remains a debilitating condition that is therapeutically challenging for clinicians. This case series identifies 23 patients with delusional infestation in an Australian setting. The majority of patients are women and unlikely to have a psychiatric comorbid background. The use of unnecessary anti-parasitic medication is prevalent.

Bacteriophage therapy for refractory Pseudomonas aeruginosa urinary tract infection.

We describe the success of adjunctive bacteriophage therapy for refractory Pseudomonas aeruginosa urinary tract infection in the context of bilateral ureteric stents and bladder ulceration, after repeated failure of antibiotics alone. No bacteriophage-resistant bacteria arose, and the kinetics of bacteriophage and bacteria in urine suggest self-sustaining and self-limiting infection.

Predictors and outcome associated with an Enterococcus positive isolate during intensive care unit admission.

This study reports the incidence, risk factors and mortality associated with a positive Enterococcus spp. isolate during admission to two tertiary intensive care units participating in an antibiotic cycling study. Incidence was low, with only 4.2% of admissions (36/852) at Royal Brisbane and Women's Hospital and 2.8% (31/1104) at Westmead Hospital developing a positive Enterococcus spp. isolate (P=0.087). A positive enterococcal isolate, while not an independent predictor of mortality (odds ratio [OR]=1.6, 95% confidence interval [CI] 0.80 to 3.2, P=0.18), may be a marker of the underlying severity of illness with higher unadjusted in-hospital mortality (26% or 17/66 vs 14% or 250/1855, P=0.007). Independent risk factors for a positive isolate were use of meropenem/imipenem (OR=5.7, 95% CI 2.4 to 14, P <0.001) and cefepime (OR=2.5, 95% CI 1.2 to 5.3, P=0.017) within 48 hours of intensive care unit admission, the presence of a nasogastric tube (OR=4.1, 95% CI 1.3 to 14, P=0.018), renal replacement therapy (OR=2.2, 95% CI 1.0 to 4.7, P=0.046), operative intervention (OR=1.8, 95% CI 1.0 to 3.2, P=0.035) and age (OR=1.2, 95% CI 1.1 to 1.5, P=0.009). None of these factors, except for the need for renal replacement therapy (OR=6.2, 95% CI 1.4 to 27, P=0.015), was associated with increased mortality. Enterococci-directed empiric therapy in the treatment of sepsis remains of unproven value, although this negative finding must be evaluated against other higher powered studies.

Carbapenem-resistant Acinetobacter baumannii in intensive care unit patients: risk factors for acquisition, infection and their consequences.

A retrospective case-control study was performed to assess risk factors and the clinical and economic consequences associated with acquisition of carbapenem-resistant Acinetobacter baumannii (CR-AB) in an intensive care unit (ICU) over a 24-month period. CR-AB was acquired by 64 of 1431 ICU admissions; each was matched with two controls. Risk factors associated with CR-AB acquisition included ICU-wide variables, such as 'colonization pressure' (the prevalence of ICU colonized patients) and ICU antibiotic use over the preceding three months, as well as patient-related variables. Among colonized patients, risk factors for CR-AB infection included transfusion and 'colonization density' (the proportion of body sites colonized with CR-AB). CR-AB infection was independently associated with increased hospital mortality [mortality difference: 20%; 95% confidence interval (CI): 1-40%], prolonged ICU stay (median length of stay difference: 15 days; 95% CI: 9-21 days) and prolonged hospital stay (30 days, 11-38 days) compared with matched controls.

Development of a real-time Staphylococcus aureus and MRSA (SAM-) PCR for routine blood culture.

The notification of "Gram-positive cocci, possibly staphylococcus" in a blood culture drawn from a seriously ill patient is responsible for a large amount of vancomycin prescribing in institutions where methicillin-resistant Staphylococcus aureus (MRSA) is an important cause of bacteraemia. A duplex real-time TaqMan polymerase chain reaction targeting the species-specific nuc gene, and the mecA gene encoding methicillin-resistance, was developed as a tool for rapid identification and detection of S. aureus and methicillin-resistance, and optimised for immediate as-needs testing. Three different DNA extraction methods achieved varying DNA quality, with PCR inhibition the main problem. Serial blood cultures (n=120) identified as possible staphylococci on Gram stain from our clinical laboratory were examined. There was one false negative result for a methicillin-resistant Staphylococcus epidermidis, which was positive on repeat testing, and one false negative result due to DNA extraction failure for MRSA from peritoneal dialysate inoculated into blood culture medium. Sensitivity and specificity of 97% and 100%, respectively, were obtained for mecA; and sensitivity and specificity of 98% and 100%, respectively, for nuc. Detection of slow-growing coagulase-negative staphylococci as co-infecting strains may be reduced. The assay quickly and reliably identified S. aureus in mixed infection, and identified methicillin resistance in both S. epidermidis and S. aureus strains.

Antimicrobial susceptibility of Bartonella henselae using Etest methodology.

OBJECTIVES: Bartonella henselae is a fastidious slow growing pathogen which is seldom cultured in the laboratory. Previous descriptions of antimicrobial susceptibility have been largely limited to feline isolates and/or laboratory reference strains, with no accounting for genotypic or phenotypic diversity. METHODS: An optimal method of antimicrobial susceptibility testing by Etest was established to compare the antimicrobial susceptibilities of 12 different isolates of B. henselae, 5 human and 7 feline, which have previously been well characterized by 16S rRNA sequencing, multi-locus sequence typing (MLST), phase variation and passage number. RESULTS: No difference in susceptibility could be attributed to differences in genotype, source of the isolate or passage number. Where comparisons were drawn with previously published results, these were found to be concordant. CONCLUSIONS: We conclude that antibiotic susceptibility can be determined by a simple Etest method for B. henselae isolates. This method is reproducible among diverse strains, and is sufficiently predictable that generalizations can be confidently made about optimal antibiotic choices.

Multiplex PCR for screening of integrons in bacterial lysates.

Bacterial integrons are a useful PCR amplification target in epidemiological surveys of bacterial antibiotic resistance, and a variety of primers have been published. We describe multiplex PCR methodology to test for classes 1, 2 and 3 integron-associated integrases in boiled lysates of Gram-negative bacteria. We report on performance in Acinetobacter spp. (n=50), Enterobacteriaceae (n=76), Pseudomonas aeruginosa (n=15), Bacteroidesspp. (n=69), and in undifferentiated mixed cultures derived from perineal swabs (n=50) and endotracheal aspirates (n=8). This method achieved 100% sensitivity and specificity in simple lysates made from a range of bacteria, without requiring DNA extraction, and is recommended as an efficient screening tool for surveys of integron cassettes.

Antibiotic resistance in the intensive care unit: a primer in bacteriology.

The clinical use of potent, well-tolerated, broad-spectrum antibiotics has been paralleled by the development of resistance in bacteria, and the prevalence of highly resistant bacteria in some intensive care units is despairingly commonplace. The intensive care community faces the realistic prospect of untreatable nosocomial infections and should be searching for new approaches to diagnose and manage resistant bacteria. In this review, we discuss some of the relevant underlying biology, with a particular focus on genetic transfer vehicles and the relationship of selection pressure to their movements. It is an attempt to demystify the relevant language and concepts for the anaesthetist and intensivist, to explain some of the reasons for the emergence of resistance in bacteria, and to provide a contextual basis for discussion of management approaches such as selective decontamination and antibiotic cycling.

Characterization of the natural population of Bartonella henselae by multilocus sequence typing.

Investigations of the population genetics of Bartonella henselae have demonstrated a high level of diversity among strains, and the delineation of isolates into one of two subtypes, type I (Houston) and type II (Marseille), represented by specific 16S ribosomal DNA (rDNA) sequences, has long been considered the most significant genotypic division within the species. This belief is challenged by recent work suggesting a role for horizontal gene exchange in generating intraspecies diversity. We attempted to resolve this issue and extend exploration of the population structure of B. henselae by using multilocus sequence typing (MLST) to examine the distribution of polymorphisms within nine different genes in a sample of 37 human and feline isolates. MLST distinguished seven sequence types (STs) that resolved into three distinct lineages, suggesting a clonal population structure for the species, and support for these divisions was obtained by macrorestriction analysis using pulsed-field gel electrophoresis. The distribution of STs among isolates recovered from human infections was not random, and such isolates were significantly more often associated with one particular ST, lending further support to the suggestion that specific genotypes contribute disproportionately to the disease burden in humans. All but one isolate lay on lineages that bore the representative strain of either the Houston or Marseille subtype. However, the distribution of the two 16S rDNA alleles among the isolates was not entirely congruent with their lineage allocations, indicating that this is not a sensitive marker of the clonal divisions within the species. The inheritances of several of the genes studied could not be reconciled with one another, providing further evidence of horizontal gene transfer among B. henselae strains and suggesting that recombination has a role in shaping the genetic character of bartonellae.

Limited diversity among human isolates of Bartonella henselae.

A study of 59 isolates of Bartonella henselae reveals relatively limited diversity among those of human origin (n = 28). Either of two distinct alleles of both gltA and 16S ribosomal DNA (rDNA) was found in all isolates, with a high level of congruity between 16S and gltA inheritance among proven human pathogens. Human isolates from all over Eastern Australia were most commonly 16S rDNA (Bergmans) type I, with the same gltA allele as the type strain (Houston-1). Comparable feline isolates were more commonly 16S type II, with less congruity of inheritance between 16S and gltA alleles. Previously described arbitrarily primed PCR and EagI-HhaI infrequent restriction site PCR fingerprinting techniques separated Bartonella species effectively but lacked discriminating power within B. henselae. Examination of the 16-23S intergenic spacer region revealed for several strains several point mutations as well as a repeat sequence of unknown significance which is readily detected by HaeIII restriction fragment length polymorphism analysis. The bacteriophage-associated papA gene was present in all isolates. Enterobacterial repetitive intergenic consensus PCR proved to be a useful and robust typing tool and clearly separated human isolates (including imported strains) from the majority of feline isolates. Our data are consistent with published evidence and with previous suggestions of intragenomic rearrangements in the type strain and suggest that human isolates come from a limited subset of B. henselae strains. They strengthen arguments for careful exploration of genotype-phenotype relationships and for the development of a multilocus enzyme electrophoresis and multilocus sequence typing-based approach to the phylogeny of B. henselae.

Non-bronchoscopic bronchoalveolar lavage in the microbiological diagnosis of pneumonia in mechanically ventilated patients.

A prospective study comparing standardized non-bronchoscopic bronchoalveolar lavage (sNB-BAL) and non-specific endotracheal aspirate (NsETA) in the microbiological diagnosis of pneumonia in mechanically ventilated patients is described. One hundred episodes in 82 mechanically ventilated patients with or without radiological and clinical diagnostic criteria of pneumonia were studied. NsETA and sNB-BAL was performed on the day of study. Fifty-one patients had pneumonia (21 ventilator-associated, 12 hospital-acquired, 18 community-acquired) and 49 had no pneumonia as defined by widely accepted clinico-radiological criteria. The sNB-BAL was found to be significantly more specific (0. 73) compared to NsETA (0.35) for the microbiological diagnosis of pneumonia. Colonization rates with NsETA were significantly higher compared to sNB-BAL (P value <0.0001). No patient had complications attributable to the sNB-BAL procedure. We conlude that sNB-BAL is a safe, effective, sensitive, specific and inexpensive procedure for the serial evaluation of pneumonia in mechanically ventilated patients.

What do physicians think about evidence-based antibiotic use in critical care? A survey of Australian intensivists and infectious disease practitioners.

BACKGROUND: The analysis of factors that influence prescribing decisions is increasingly important. Antibiotic use is often based on limited evidence and lack of information about clinical decision-making processes is an important obstacle to improving antibiotic utilization. AIMS: To compare the attitudes of intensive care unit practitioners (ICUP) and infectious disease practitioners (IDP) to antibiotic use and to the evidence-based information support. METHOD: A postal survey conducted between March and July 2000 of ICUP and IDP representing all States and Territories in Australia. RESULTS: One hundred and fifty-three of 224 clinicians returned the questionnaire (68.3% response rate). In choosing an antibiotic, IDP placed significantly more weight than ICUP on the in vitro susceptibility of the pathogen (P = 0.001), antibiotic cost (P = 0.05) and possible development of antibiotic resistance (P = 0.007). More than 95% of both groups believed that unit-specific antibiotic susceptibility of endemic pathogens was an essential factor in rational prescribing, but only 68.5% of IDP and 38.7% of ICUP use microbiology laboratory databases. When in doubt about appropriate antibiotic use, 63.8% of ICUP seek and 76.3% usually follow the advice of IDP. Both groups agree that published antibiotic guidelines are useful, but IDP were more likely to consult them. ICUP were more likely to believe that guidelines are used to control clinicians rather than to improve quality of care (P = 0.001). A greater proportion of IDP (71.2%) than ICUP (52.5%) believed that antibiotic prescribing in their intensive care unit (ICU) was evidence based but most (91.8% and 86.9%, respectively) agreed that it should be. CONCLUSIONS: Australian clinicians have positive views about evidence-based prescribing and antibiotic guidelines. However, there are clinically significant differences in prescribing behaviour between ICUP and IDP. These may be explained by different disease spectra managed by each group or different cultures, training and/or cognitive styles. Improvements in the understanding of physicians' information and decision support needs are required to strengthen evidence-based prescribing.