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.

Urinary piperacillin/tazobactam pharmacokinetics in vitro to determine the pharmacodynamic breakpoint for resistant Enterobacteriaceae.

Urinary tract infections caused by multidrug-resistant Enterobacteriaceae are a growing burden worldwide. Recent studies of urinary pharmacokinetics described high piperacillin/tazobactam (TZP) concentrations in urine, but it is unknown whether this results in treatment efficacy. This study investigated the pharmacodynamics of TZP in a static in vitro model for Enterobacteriaceae to determine the concentration-effect relationship and ultimately the required free (unbound) time above the minimum inhibitory concentration (fT>MIC) required for bacterial killing. The static simulation model investigated TZP fT>MIC between 0% and 100%. Resistant Escherichia coli and Klebsiella pneumoniae isolates with piperacillin/tazobactam MICs of 4096/512, 1024/128 and 128/16 mg/L were investigated; two of the three organisms were carbapenemase-producers. Clinical efficacy was determined as a 3-log reduction over the dosing interval by comparing interval growth with controls. TZP was observed to exhibit time dependence for all organisms. The fT>MIC was determined to be 37.5%, 37.5% and 50% for MICs of 4096/512, 1024/128 and 128/16 mg/L, respectively. Linear regression identified the overall target to be 49.85 ± 16.9% fT>MIC. In conclusion, bactericidal activity against TZP-resistant Enterobacteriaceae occurred at 49.85 ± 16.9% fT>MIC. This suggests that highly resistant urinary organisms, including carbapenemase-producers, with MICs up to 4096/512 mg/L could be treated with TZP. Further investigations are required to elucidate urinary breakpoints and to explore the impact of different resistance mechanisms.

Survey and genetic characterization of Vibrio cholerae in Apalachicola Bay, Florida (2012-2014).

AIMS: A small outbreak of gastroenteritis in 2011 in Apalachicola Bay, FL was attributed to consumption of raw oysters carrying Vibrio cholerae serotype O75. To better understand possible health risks, V. cholerae was surveyed in oysters, fish and seawater, and results were compared to data for Vibrio vulnificus and Vibrio parahaemolyticus. METHODS AND RESULTS: Enrichment protocols were used to compare prevalence of V. cholerae (0, 48, 50%), V. vulnificus (89, 97, 100%) and V. parahaemolyticus (83, 83, 100%) in fish, seawater and oysters respectively. Compared to other species, Most probable number results indicated significantly (P < 0·001) lower abundance of V. cholerae, which was also detected more frequently at lower salinity, near-shore sites; other species were more widely distributed throughout the bay. Genes for expression (ctxA, ctxB) and acquisition (tcpA) of cholera toxin were absent in all strains by PCR, which was confirmed by whole genome sequencing; however, other putative virulence genes (toxR, rtxA, hlyA, opmU) were common. Multi-locus sequence typing revealed 78% of isolates were genetically closer to V. cholerae O75 lineage or other non-O1 serogroups than to O1 or O139 serogroups. Resistance to amoxicillin, kanamycin, streptomycin, amikacin, tetracycline and cephalothin, as well as multidrug resistance, was noted. CONCLUSIONS: Results indicated minimal human health risk posed by V. cholerae, as all isolates recovered from Apalachicola Bay did not have the genetic capacity to produce cholera toxin. Vibrio cholerae was less prevalent and abundant relative to other pathogenic Vibrio species. SIGNIFICANCE AND IMPACT OF THE STUDY: These studies provide important baseline observations for V. cholerae virulence potential regarding: (i) genetic relatedness to V. cholerae O75, (ii) antibiotic resistance and (iii) prevalence of multiple virulence genes. These data will serve as a biomonitoring tool to better understand ecosystem status and management if bacterial densities and virulence potential are altered by environmental and climatic changes over time.

Application of chitosan microparticles for mitigation of Salmonella in agricultural water.

AIM: The activity of chitosan microparticles (CM) was examined using a matrix of conditions in order to assess the efficacy of CM as a mitigation against various strains of Salmonella enterica in agricultural water. METHODS AND RESULTS: Different concentrations of CM (0, 0·01, 0·1, 0·2, 0·3% w/v) were examined for antimicrobial activity against log vs stationary phase cells of Salmonella and at different conditions of temperature, salinity and pH. Results showed greatest activity with 0·3% CM at pH 7, 25-37°C without additional of salt. Significant reductions in Salmonella levels were also achieved in natural pond water, although decreases were reduced compared to sterile water. All serotypes were sensitive to CM, with minimal inhibitory concentrations ranging from 0·0031 to 0·0250% w/v. Phylogenic analysis of Javiana strains showed increased resistance appeared in multiple genetic lineages. CONCLUSION: Conditions demonstrating greatest CM activity were compatible with agricultural practices. Although sensitivity to CM varied among Salmonella strains, all strains were sensitive under conditions examined in this study. SIGNIFICANCE AND IMPACT OF THE STUDY: This research indicated that CM, a natural compound with minimal environmental impact, could be an effective alternative for mitigating Salmonella in agricultural water applications.

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.

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.

Detection and identification of carprofen and its in vivo metabolites in greyhound urine by capillary gas chromatography-mass spectrometry.

Rimadyl (carprofen) was administered orally to the racing greyhound at a dose of 2.2 mg kg(-1). Following both alkaline and enzymatic hydrolysis, postadministration urine samples were extracted by mixed mode solid-phase extraction (SPE) cartridges to identify target analyte(s) for forensic screening and confirmatory analysis methods. The acidic isolates were derivatised as trimethylsilyl ethers (TMS) and analysed by gas chromatography-mass spectrometry (GC-MS). Carprofen and five phase I metabolites were identified. Positive ion electron ionisation (EI(+)) mass spectra of the TMS derivatives of carprofen and its metabolites show a diagnostic base peak at M(+)*. -117 corresponding to the loss of COO-Si-(CH(3))(3) group as a radical. GC-MS with positive ion ammonia chemical ionisation (CI(+)) of the compounds provided both derivatised molecular mass and some structural information. Deutromethylation-TMS derivatisation was used to distinguish between aromatic and aliphatic oxidations of carprofen. The drug is rapidly absorbed, extensively metabolised and excreted as phase II conjugates in urine. Carprofen, three aromatic hydroxy and a minor N-hydroxy metabolite were detected for up to 48 h. For samples collected between 2 and 8 h after administration, the concentration of total carprofen ranged between 200 and 490 ng ml(-1). The major metabolite, alpha-hydroxycarprofen was detected for over 72 h and therefore can also be used as a marker for the forensic screening of carprofen in greyhound urine.

Heliox improves pulmonary mechanics in a pediatric porcine model of induced severe bronchospasm and independent lung mechanical ventilation.

BACKGROUND: A helium-oxygen gas mixture (heliox) has low gas density and low turbulence and resistance through narrowed airways. The effects of heliox on pulmonary mechanics following severe methacholine-induced bronchospasm were investigated and compared to those of a nitrogen-oxygen gas mixture (nitrox) in an innovative pediatric porcine, independent lung, mechanical ventilation model. RESULTS: All of the lungs showed evidence of severe bronchospasm after methacholine challenge. Prospective definition of 'heliox response' was a 15% or greater improvement in lung function in the lung receiving heliox compared with the matched lung receiving nitrox. Seven out of 10 pigs responded to heliox therapy with respect to resistance and eight out of 10 pigs responded to heliox therapy with respect to compliance and tidal volume (P < 0.03). After crossover from nitrox to heliox, eight out of eight lungs significantly improved with respect to tidal volume, resistance and compliance (P < 0.001). After crossover from heliox to nitrox all eight lungs showed a significant increase in resistance and a significant decrease in tidal volume (P < 0.001). CONCLUSIONS: In a pediatric porcine model of acute, severe methacholine-induced bronchospasm and independent lung mechanical ventilation, administration of heliox improves pulmonary mechanics, gas flow, and ventilation. Administration of heliox should be considered for support of pediatric patients with acute, severe bronchospasm requiring mechanical ventilation through small artificial airways.

Anesthetic regimen effects on a pediatric porcine model of asphyxial arrest.

The effects of three anesthetic regimens on an established model of pediatric porcine hypoxic-hypercarbic arrest were examined. Twenty-four preadolescent miniature piglets were paralyzed, mechanically ventilated and anesthetized with one of three regimens: IM + IV pentobarbital (n = 8); IM + IV ketamine (n = 8); or IM ketamine+inhaled isoflurane (n = 8). Asphyxial cardiopulmonary arrest was induced and, after and 8 min cardiac arrest nonintervention interval, a standardized protocol of manual CPR with mechanical ventilation was performed. Outcome variables included incidence of ventricular fibrillation, time to cardiac arrest, endogenous plasma epinephrine levels and arteriovenous epinephrine gradients. IV Ketamine anesthesia produced the highest incidence of ventricular fibrillation (P < 0.01 vs. pentobarbital and isoflurane). Time to asphyxia induced cardiac arrest was greatest for the pentobarbital group (P < 0.05 vs. ketamine and isoflurane). During induction of asphyxial cardiac arrest (low cardiac flow), endogenous venous epinephrine accumulation was highest in the pentobarbital anesthetized group (P < 0.05). After 8 min of untreated cardiac arrest and 1 min of CPR (low flow), arterial epinephrine levels were highest in the ketamine group (P < 0.05). Endogenous epinephrine gradients were venous > arterial in all groups at the end of the 8 min cardiac arrest non-intervention interval (no flow). After 1 min of CPR, the gradients had either equalized or reversed to arterial > venous in all groups except for pentobarbital. As designed and expected, return of spontaneous circulation did not occur in any animal. We conclude that, in developing models of porcine asphyxial cardiopulmonary arrest and resuscitation to simulate pediatric human arrest, variations in anesthetic regimen produce significant differences in parameters that are important to consider: time to asphyxia induced cardiac arrest, fibrillation threshold, plasma epinephrine level and arteriovenous epinephrine gradient. Anesthetic effects need to be carefully considered and clearly explained to facilitate the interpretation of studies of interventions in cardiopulmonary arrest and resuscitation.

Cortisol concentrations in post competition horse urine: a French and British survey.

The purpose of the present report was to estimate the population parameters of cortisol concentrations in urine, an endogenous hormone used as a 'doping' agent and for which an international threshold (1.0 micrograms/ml) has been proposed. Two data bases (French and UK) corresponding to 112 and 142 samples, respectively were considered. Urine was collected under specific post competition conditions. Cortisol concentrations were obtained by validated methods (HPLC for the French samples, and GC-MS for UK samples). No difference was observed between the 2 data sets and statistical analyses were carried out on the two merged files. The overall geometric mean cortisol concentration was 48 ng/ml. Distribution was not Gaussian. A log-normal distribution was not rejected (for P > 0.05). Using the log-normal distribution, it was calculated that the probability of exceeding a cortisol concentration in urine of 1.0 micrograms/ml was 1.1 x 10(-4). It was concluded that the actual international threshold is specific i.e. robust with regard to the risk of erroneously declaring an unmedicated horse as positive.

Comparison of the use of mass spectrometry and methylene unit values in the determination of the stereochemistry of estranediol, the major urinary metabolite of 19-nortestosterone in the horse.

The stereochemistry of an isomer of 5-estrane-3,17 alpha-diol, the major metabolite of 19-nortestosterone in horse urine has been established by the use of methylene unit (MU) values. The empirical MU values of the bis-trimethylsilyl (TMS) derivatives of the eight available isomers of 5-androstane-3,17-diol and four isomers of 5-estrane-3,17 beta-diol were determined by capillary gas chromatography using three different columns. From this data the theoretical MU values for the bis-TMS derivatives of the four 5-estrane-3,17 alpha-diol isomers were predicted. Comparison of the experimentally determined MU value of the urinary metabolite with those of the theoretical values established the correct stereochemistry of the steroid. This method has been compared with the use of gas chromatography-mas spectrometry in the determination of the stereochemistry of unknown metabolites.