More than just the gene: Investigating expression using a non-native plasmid and host and its impact on resistance conferred by ß-lactamase OXA-58 isolated from a hospital wastewater microbiome.

With the escalation of hospital-acquired infections by multi-drug resistant bacteria, understanding antibiotic resistance is of paramount importance. This study focuses on the ß-lactamase gene, blaOXA-58, an important resistance determinant identified in a patient-facing hospital wastewater system. This study aimed to characterise the behaviour of the OXA-58 enzyme when expressed using a non-native plasmid and expression host. blaOXA-58 was cloned using a pET28a(+)/Escherichia coli BL21(DE3) expression system. Nitrocefin hydrolysis and antimicrobial susceptibility of OXA-58-producing cells were assessed against penicillin G, ampicillin, meropenem, and amoxicillin. blaOXA-58 conferred resistance to amoxicillin, penicillin G, and ampicillin, but not to meropenem. This was unexpected given OXA-58's annotation as a carbapenemase. The presence of meropenem also reduced nitrocefin hydrolysis, suggesting it acts as a competitive inhibitor of the OXA-58 enzyme. This study elucidates the phenotypic resistance conferred by an antimicrobial resistance gene (ARG) obtained from a clinically relevant setting and reveals that successful functional expression of ARGs is multifaceted. This study challenges the reliability of predicting antimicrobial resistance based solely on gene sequence alone, and serves as a reminder of the intricate interplay between genetics and structural factors in understanding resistance profiles across different host environments.

Acinetobacter baumannii biofilm biomass mediates tolerance to cold plasma.

Acinetobacter baumannii is an intrinsically multidrug-resistant pathogen that, when existing as a biofilm, confers increased environmental tolerance to desiccation, nutrient starvation as well as increased tolerance to antimicrobials. Outbreaks of A. baumannii infections within the clinical setting are often associated with the biofilm phenotype. This study investigates the role of biofilm biomass in A. baumannii susceptibility to exposure to a kilohertz-driven, in-house-designed, cold plasma jet, through the examination of cold plasma treatment efficacy in A. baumannii biofilms grown over various times for up to 72 h. For biofilms grown for 24, 48 and 72 h, D values were 19·32 ± 2·71, 29·18 ± 3·15 and 24·70 ± 3·07 s respectively. Monitoring A. baumannii biofilm biomass over these time periods revealed that the greatest biomass was observed at 48 h with the lowest biofilm biomass at 24 h growth. Enumeration of viable biofilm colony counts at each time point was comparable. Scanning electron microscopy images of plasma-treated biofilms revealed extensive surface damage of A. baumannii cells. These results describe the role of biomass in mediating A. baumannii biofilm susceptibility to cold plasma treatment, implicating the biofilm matrix as a protective barrier to the antimicrobial effects of cold plasma. SIGNIFICANCE AND IMPACT OF THE STUDY: Acinetobacter baumannii biofilm formation results in increased environmental and antimicrobial tolerance and resistance compared to the planktonic phenotype. Cold plasma technology is increasingly investigated as a new tool for decontamination of biofilm-contaminated surfaces, especially those found in the clinical setting. This new technology presents a promising approach to the remediation of surfaces contaminated by biofilms. This study identifies the role played by A. baumannii biofilm biomass in mediating tolerance and susceptibility to cold plasma treatment. This work demonstrates that increased biofilm biomass reduces the efficacy of antimicrobial species generated by cold plasma, resulting in greater tolerance to plasma exposure.

Microbiological interactions with cold plasma.

There is a diverse range of microbiological challenges facing the food, healthcare and clinical sectors. The increasing and pervasive resistance to broad-spectrum antibiotics and health-related concerns with many biocidal agents drives research for novel and complementary antimicrobial approaches. Biofilms display increased mechanical and antimicrobial stability and are the subject of extensive research. Cold plasmas (CP) have rapidly evolved as a technology for microbial decontamination, wound healing and cancer treatment, owing to the chemical and bio-active radicals generated known collectively as reactive oxygen and nitrogen species. This review outlines the basics of CP technology and discusses the interactions with a range of microbiological targets. Advances in mechanistic insights are presented and applications to food and clinical issues are discussed. The possibility of tailoring CP to control specific microbiological challenges is apparent. This review focuses on microbiological issues in relation to food- and healthcare-associated human infections, the role of CP in their elimination and the current status of plasma mechanisms of action.

A novel characterized multi-drug-resistant Pseudocitrobacter sp. isolated from a patient colonized while admitted to a tertiary teaching hospital.

BACKGROUND: Reports of nosocomial infections typically describe recognised microorganisms. Here, a novel bacterial species was isolated, based on rectal swab screening for carbapenemases post-admission, then phenotypically and genetically characterized. METHODS: Sensititre, Vitek and API kits, MALDI and Illumina MiSeq were employed before profiles and phylogeny were compared with other related species. FINDINGS: Determined to be a possible Enterobacterales, the isolate was found to have 99.7% 16s rRNA identity to Pseudocitrobacter corydidari; an Asian cockroach-associated species. Given the highly conserved/low variability of 16S rRNA genes in Enterobacterales, average nucleotide identity (ANI) analysis compared the new isolate's genome with those of 18 Enterobacteriaceae species, including confirmed species of Pseudocitrobacter and unnamed Pseudocitrobacter species in the SILVA database. Of these, Pseudocitrobactercorydidari had the highest ANI at 0.9562. The published genome of the only known isolate of P.corydidari does not include Antimicrobial Resistance Genes (ARGs), with exception of potential drug efflux transporters. In contrast, our clinical isolate bears recognised antimicrobial resistance genes, including Klebsiella pneumoniae carbapenemase. The associated genome suggests resistance to carbapenems, ß-lactams, sulfonamides, fluoroquinolones, macrolides, aminoglycosides and cephalosporins. Phenotypic antimicrobial resistance was confirmed. CONCLUSION: Evident variations in ARG profiles, human colonization and origin in a clinically relevant niche that is geographically, physically and chemically disparate lend credibility for divergent evolution or, less likely, parallel evolution with P. corydidari. Genome data for this new species have been submitted to GENBANK using the proposed nomenclature Pseudocitrobacter limerickensis. The patient was colonized, rather than infected, and did not require antimicrobial treatment.

Microbiome-derived antimicrobial peptides show therapeutic activity against the critically important priority pathogen, Acinetobacter baumannii.

Acinetobacter baumannii is designated by the World Health Organisation as a critical priority pathogen. Previously we discovered antimicrobial peptides (AMPs), namely Lynronne-1, -2 and -3, with efficacy against bacterial pathogens, such as Staphylococcus aureus and Pseudomonas aeruginosa. Here we assessed Lynronne-1, -2 and -3 structure by circular dichroism and efficacy against clinical strains of A. baumannii. All Lynronne AMPs demonstrated alpha-helical secondary structures and had antimicrobial activity towards all tested strains of A. baumannii (Minimum Inhibitory Concentrations 2-128 µg/ml), whilst also having anti-biofilm activity. Lynronne-2 and -3 demonstrated additive effects with amoxicillin and erythromycin, and synergy with gentamicin. The AMPs demonstrated little toxicity towards mammalian cell lines or Galleria mellonella. Fluorescence-based assay data demonstrated that Lynronne-1 and -3 had higher membrane-destabilising action against A. baumannii in comparison with Lynronne-2, which was corroborated by transcriptomic analysis. For the first time, we demonstrate the therapeutic activity of Lynronne AMPs against A. baumannii.

Atmospheric pressure, nonthermal plasma inactivation of MS2 bacteriophage: effect of oxygen concentration on virucidal activity.

AIMS: The main aim of this study was to determine the virucidal inactivation efficacy of an in-house-designed atmospheric pressure, nonthermal plasma jet operated at varying helium/oxygen feed gas concentrations against MS2 bacteriophage, widely employed as a convenient surrogate for human norovirus. METHODS AND RESULTS: The effect of variation of percentage oxygen concentration in the helium (He) carrier gas was studied and found to positively correlate with MS2 inactivation rate, indicating a role for reactive oxygen species (ROS) in viral inactivation. The inactivation rate constant increased with increasing oxygen concentrations up to 0·75% O2 . 3 log10 (99·9%) reductions in MS2 viability were achieved after 3 min of exposure to the plasma source operated in a helium/oxygen (99·25% : 0·75%) gas mixture, with >7 log10 reduction after 9 min exposure. CONCLUSIONS: Atmospheric pressure, nonthermal plasmas may have utility in the rapid disinfection of virally contaminated surfaces for infection control applications. SIGNIFICANCE AND IMPACT OF STUDY: The atmospheric pressure, nonthermal plasma jet employed in this study exhibits rapid virucidal activity against a norovirus surrogate virus, the MS2 bacteriophage, which is superior to previously published inactivation rates for chemical disinfectants.

Large-scale characterization of hospital wastewater system microbiomes and clinical isolates from infected patients: profiling of multi-drug-resistant microbial species.

BACKGROUND: Hospital-acquired infections (HAIs) and infectious agents exhibiting antimicrobial resistance (AMR) are challenges globally. Environmental patient-facing wastewater apparatus including handwashing sinks, showers and toilets are increasingly identified as sources of infectious agents and AMR genes. AIM: To provide large-scale metagenomics analysis of wastewater systems in a large teaching hospital in the Republic of Ireland experiencing multi-drug-resistant HAI outbreaks. METHODS: Wastewater pipe sections (N=20) were removed immediately prior to refurbishment of a medical ward where HAIs had been endemic. These comprised toilet U-bends, and sink and shower drains. Following DNA extraction, each pipe section underwent metagenomic analysis. FINDINGS: Diverse taxonomic and resistome profiles were observed, with members of phyla Proteobacteria and Actinobacteria dominating (38.23 ± 5.68% and 15.78 ± 3.53%, respectively). Genomes of five clinical isolates were analysed. These AMR bacterial isolates were from patients >48 h post-admission to the ward. Genomic analysis determined that the isolates bore a high number of antimicrobial resistance genes (ARGs). CONCLUSION: Comparison of resistome profiles of isolates and wastewater metagenomes revealed high degrees of similarity, with many identical ARGs shared, suggesting probable acquisition post-admission. The highest numbers of ARGs observed were those encoding resistance to clinically significant and commonly used antibiotic classes. Average nucleotide identity analysis confirmed the presence of highly similar or identical genomes in clinical isolates and wastewater pipes. These unique large-scale analyses reinforce the need for regular cleaning and decontamination of patient-facing hospital wastewater pipes and effective infection control policies to prevent transmission of nosocomial infection and emergence of AMR within potential wastewater reservoirs.

Comparison of the cidal activity of tea tree oil and terpinen-4-ol against clinical bacterial skin isolates and human fibroblast cells.

AIMS: The aim of this study was to compare both the antimicrobial activity of terpinen-4-ol and tea tree oil (TTO) against clinical skin isolates of meticillin-resistant Staphylococcus aureus (MRSA) and coagulase-negative staphylococci (CoNS) and their toxicity against human fibroblast cells. METHODS AND RESULTS: Antimicrobial activity was compared by using broth microdilution and quantitative in vitro time-kill test methods. Terpinen-4-ol exhibited significantly greater bacteriostatic and bactericidal activity, as measured by minimum inhibitory and bactericidal concentrations, respectively, than TTO against both MRSA and CoNS isolates. Although not statistically significant, time-kill studies also clearly showed that terpinen-4-ol exhibited greater antimicrobial activity than TTO. Comparison of the toxicity of terpinen-4-ol and TTO against human fibroblasts revealed that neither agent, at the concentrations tested, were toxic over the 24-h test period. CONCLUSIONS: Terpinen-4-ol is a more potent antibacterial agent against MRSA and CoNS isolates than TTO with neither agent exhibiting toxicity to fibroblast cells at the concentrations tested. SIGNIFICANCE AND IMPACT OF THE STUDY: Terpinen-4-ol should be considered for inclusion as a single agent in products formulated for topical treatment of MRSA infection. However, further work would initially be required to ensure that resistance would not develop with the use of terpinen-4-ol as a single agent.

Robot-assisted repair of diaphragmatic hernias following ventricular assist device implantation.

Use of ventricular assist devices (VADs) is increasingly common, as is the need for surgeons to be familiar with the management of common complications in this population. Nonetheless, repair of diaphragmatic hernias which commonly develop following VAD implantation remains technically challenging due to intra-abdominal adhesions and the proximity of vital structures. Despite the potential benefits of improved dexterity and visualization, robotic approaches have thus far not been used to address this. We present the first two described cases of robot-assisted repair of diaphragmatic hernias in the setting of prior or current VAD placement.

Antimicrobial efficacy of an innovative emulsion of medium chain triglycerides against canine and feline periodontopathogens.

OBJECTIVES: To test the in vitro antimicrobial efficacy of a non-toxic emulsion of free fatty acids against clinically relevant canine and feline periodontopathogens METHODS: Antimicrobial kill kinetics were established utilising an alamarBlue(®) viability assay against 10 species of canine and feline periodontopathogens in the biofilm mode of growth at a concentration of 0·125% v/v medium chain triglyceride (ML:8) emulsion. The results were compared with 0·12% v/v chlorhexidine digluconate and a xylitol-containing dental formulation. Mammalian cellular cytotoxicity was also investigated for both the ML:8 emulsion and chlorhexidine digluconate (0·25 to 0·0625% v/v) using in vitro tissue culture techniques. RESULTS: No statistically significant difference was observed in the antimicrobial activity of the ML:8 emulsion and chlorhexidine digluconate; a high percentage kill rate (>70%) was achieved within 5 minutes of exposure and was maintained at subsequent time points. A statistically significant improvement in antibiofilm activity was observed with the ML:8 emulsion compared with the xylitol-containing formulation. The ML:8 emulsion possessed a significantly lower (P < 0·001) toxicity profile compared with the chlorhexidine digluconate in mammalian cellular cytotoxicity assays. CLINICAL SIGNIFICANCE: The ML:8 emulsion exhibited significant potential as a putative effective antimicrobial alternative to chlorhexidine- and xylitol- based products for the reduction of canine and feline periodontopathogens.

Quality control of agar diffusion susceptibility tests. Data from the Quality Assurance Service Microbiology Program of the College of American Pathologists.

During the period from January 1979 through January 1981, 155 active participants in the Microbiology Program of the College of American Pathologists Quality Assurance Service (QAS) submitted approximately two million individuals determinations on three quality control reference strains. Of these determinations, 83% were obtained using the standardized disc-diffusion procedure of Bauer and co-workers, and, 6% using the agar overlay modification of Barry and associates. The mean and standard deviation for data obtained using these procedures were similar to those for data collected from the inception of the program in 1974 through December 1978. The number of individual determinations falling above or below existing control limits, however, was significantly less for the present data than for that previously reported. These differences result from a change in computation procedures. In the current report percentages of daily values exceeding the NCCLS limits were calculated using actual counts, whereas in the previous reports these percentages were estimated by accepting an assumption of Gaussian distribution.

Contemporary quality control practices for antimicrobial susceptibility tests: a report from the microbiology portion of the College of American Pathologists (CAP) surveys program.

The evaluation of quality control (QC) practices from the College of American Pathologists (CAP) Microbiology Surveys demonstrates a trend away from daily controls similar to that first noted in 1980. Previous recommendations for more relevant cost-containment oriented weekly QC procedures are discussed, and the 1982 CAP Surveys statistics confirm earlier comparability between the daily and weekly QC participant groups. Each QC practice subgroup (daily or weekly) had nearly identical mean disk inhibitory zone diameters, and the performance accuracy on two 1982 challenge strains were very similar. Generally, disk zone diameter from those laboratories using weekly QC were smaller, but the frequency of minor and major (false-resistant) errors was only slightly increased. The authors recommend the conversion to weekly QC of disk or dilution antimicrobial susceptibility tests after an acceptable daily QC performance has been established (See criteria in discussion).

Interlaboratory survey of enzyme analyses: V. A simplified method of reporting participants' data.

The College of American Pathologists' Enzyme Survey continues to provide a valuable measure of the analytic quality of clinical enzymology for its participants and for the laboratory community. An updated reporting format for the participants simplifies the interpretation of Enzyme Survey data and should make it more valuable for detecting short- and long-term changes in accuracy and precision. A short-term CV of 3% and a long-term CV of 6% are recommended as analytic goals for the five enzymes described here. The upper limit of normal used by many laboratories is appropriate for a given enzyme yet remains as an area requiring urgent further study.

Stability of sodium and potassium in lyophilized quality control serum. A study of data from the Quality Assurance Service Program of the College of American Pathologists.

The long-term prereconstitution stability of sodium and potassium in large pools of lyophilized quality control serum used in Regional Quality Control Programs, conducted between 1977 and 1980 is reviewed. In approximately one third of 27 pools studied, minimal but significant increases in sodium concentration were detected, these increases averaged less than 0.50 per year and confirmed our previously reported results from pools analyzed between 1973 and 1976. Results relating to potassium stability have been inconsistent. The previously reported tendency for potassium to increase in some pools was again suggested by data obtained from laboratories using automated flame-emission photometry procedures. This was not confirmed, however, by the data reflecting manual/semi-automated flame-emission photometry procedures or automated electrode methods. It is postulated, that the source of sodium causing the increases in concentration is the glass containers in which control materials are stored between the time of manufacture and reconstitution. Regression analysis against time of monthly interlaboratory means, from participants in Regional Quality Control Programs, is a useful tool for evaluating postmanufacture stability of analytes in pools used for daily internal quality control procedures.

Glucose stability in lyophilized chemistry quality control serum. A study of data from the quality assurance service (QAS) program of the College of American Pathologists.

Data from 2.5 million glucose analyses on pools of lyophilized human quality control serum were used to evaluate analyte stability in the prereconstitution phase. Input information was from laboratories in Regional Quality Control Programs that use CAP Quality Assurance Service (QAS) data processing. Of 31 pools in use between 1977 and 1981, decreasing glucose concentration was detected by, at least, one method in 26 pools, and by two or more methods in 21 pools. Method-associated average decrease in concentration varied from 0.13 mg/dL/month (glucose oxidase-electrode) to 0.19 mg/dL/month (automated glucose oxidase-colorimetric). Bidirectional instability as a function of analytic method, i.e., increase with "mild" methods, decrease with "rigorous" methods that was noticed previously with pools analyzed between 1973 and 1977, was no longer seen. Dominant directional changes in the later pools were downward by all methods, when statistically significant trends were demonstrated.

Analytic clinical laboratory precision. State of the art for selected enzymes.

Results of precision estimates are reported for selected enzyme analytes. Laboratories that participated in regional quality control programs supplied data to a centralized data processing service (College of American Pathologists Computer Center). These data, representing several regional groups which used different commercial sources of control serum, were statistically analyzed by regression of coefficient of variation (CV) upon concentration of analyte using a modified polynomial regression technic. Tolerance limits for long-term within-laboratory precision were developed from this data. Estimated CVs were extracted at selected analyte concentrations, and tolerance limits for CVs at these concentrations were developed. In addition, selected individual methods within the larger groups were individually examined to evaluate effect of control material matrix upon clinical estimates of enzyme precision. Differences in selected method groups are discussed.

Stability of mean assay values of magnesium and iron in lyophilized quality control serum: a study based on data from the quality assurance service (QAS) of the College of American Pathologists.

Data from magnesium and iron analyses of pools of lyophilized quality control serum were used to evaluate stability of analyte mean values in the pre-reconstitution period. Information was received from laboratories in Regional Quality Control Programs between 1977 and 1981, using CAP Quality Assurance Service data processing. For magnesium, 28 of 41 (68%) pool-method combinations were stable, 11 (27%) showed decreases, and 2 (5%) showed increases. Decreases averaged 0.008 mg/dL/month (all methods). A pronounced effect of method (automated methylthymol blue) and year correlated with decreases in measurable magnesium. For iron, 45 of 56 (80%) pool-method combinations were stable, 8 (15%) showed decreases, and 3 (5%) showed increases. Decreases averaged 0.349 g/dL (all methods). In most cases, changes in measured concentrations are attributed to methodologic factors, rather than intrinsic changes in analyte concentrations.

Multiprogram characterization of laboratory bias, precision, and total error. Proposal for improved assessment with shared external and expanded internal (regional) quality control pools.

We establish that, for the analytes aspartate aminotransferase, glucose, phosphorus, and potassium, there is significant correlation between laboratory performance as determined by College of American Pathologists-sponsored external (Surveys) and expanded internal (regional) quality control (Quality Assurance Service) programs. However, relatively low parametric and nonparametric correlation coefficients and significant departure of linear regression slopes from unity reflect major differences in the calculated parameters of absolute bias, precision, and total error obtained through internal and external quality control. Significantly better performance in both Surveys and the Quality Assurance Service was documented for laboratories participating in the College of American Pathologists Laboratory Accreditation Program. Multiple descriptors of laboratory quality, as indicated here, are superior in describing laboratory performance to proficiency testing alone. As a bridge between external and internal quality control, shared pools of quality control materials are described for use as unknown Survey challenges and subsequent distribution for regional quality control. Such programs, which can be configured to serve thousands of laboratories, could offer cost savings, better quality assurance, and improved characterization of laboratory performance by minimizing interprogram differences in control matrix and method classification and providing greater reliability of target values.

Matrix effects and the accuracy of cholesterol analysis.

We found evidence of bias due to matrix effect in 70% of 37 instrument/reagent-specific systems analyzing the total cholesterol content of a lyophilized proficiency testing material. We used a computational method to remove bias due to matrix effect from the proficiency testing database. After correction for matrix effect bias and when compared with the reference method, 92% to 93% of results for three lyophilized proficiency testing samples analyzed in 1989 and 1990 met the 1992 National Cholesterol Education Program total error goal of 8.9%, and 94% to 95% met the Clinical Laboratory Improvement Amendments of 1988 (CLIA '88) goal of 10%. However, compared with the definitive method for total cholesterol, the calibration bias of 41% of 37 peer groups exceeded the 1992 National Cholesterol Education Program goal for bias of 3%. Because the calibration bias of the method is incorporated into the peer group mean, use of peer group means as target values to assess result acceptability hinders advancement of the state of the art in interlaboratory comparability and the clinical effectiveness of laboratory testing. The prevalence of matrix effects has prevented successful application of accuracy-based evaluation of cholesterol test proficiency. The establishment of predictable recovery, preferably complete recovery, of cholesterol from reference materials is an important priority for cholesterol test methods. However, adjustment of proficiency testing results to remove the average bias due to matrix effects can help assess the actual state of the art in cholesterol test accuracy.

Peptides containing acylated C-terminal gem diamines: novel irreversible inactivators of the cysteine and serine proteinases.

This study reports on the synthesis of peptides containing C-terminal acylated gem-diamines and their utilization for the preparation of irreversible inactivators of the serine and cysteine proteinases. We have succeeded in obtaining an inhibitor Acetyl-Val-Pro-g-Val-CO-O-C(6)H(4)-NO(2) of neutrophil and pancreatic elastases that functions in a time-dependent manner, indicative of the action of an irreversible inactivator, functioning, most probably, through the formation of a long-lived acyl enzyme intermediate. In addition, we have demonstrated the irreversible inhibition of the cysteine proteinase bovine cathepsin B, by chloroacetyl and bromoacetyl derivatives of a dipeptide gem-diamine, Cbz-Phe-g-Ala-CO-CH(2)Hal (Hal = Br, Cl).