Specific discrimination of pathogenic bacteria causing septic arthritis using Raman spectroscopy: In-vitro study.

In this study we performed preliminary experiments using Raman spectroscopy as an evolving technology in biofluid and microbial characterization, to explore its potential for rapid diagnosis of pathogenic bacteria in an in-vitro synovial fluid infection model. Normal human synovial fluids samples were collected from patients undergoing knee surgery and the three most common pathogenic bacteria introduced in-vitro into the samples. The bacterial growth was systematically monitored using a Raman spectroscopy. Multivariate regression analysis of acquired spectra showed bacterial characteristic Raman bands related to bacterial cell membranes and DNA structures to increase continuously as the incubation period was increased. Spectra signature recorded from cultured synovial fluid samples showed a significant loss in synovial quality and protein morphology over time compared to control samples. In this study, Raman spectroscopy shows promise for rapid pathogenic bacteria identification in synovial fluid. Marker peaks distinguished inoculated bacteria, while chemical changes reveal infection dynamics.

Hypervirulent Capsular Serotypes K1 and K2 Klebsiella pneumoniae Strains Demonstrate Resistance to Serum Bactericidal Activity and Galleria mellonella Lethality.

Hypervirulent Klebsiella pneumoniae (hvKp) is a variant that has been increasingly linked to severe, life-threatening infections including pyogenic liver abscess and bloodstream infections. HvKps belonging to the capsular serotypes K1 and K2 have been reported worldwide, however, very scarce studies are available on their genomics and virulence. In the current study, we report four hypermucoviscous extended-spectrum ß-lactamase-producing hvKp clinical strains of capsular serotype K1 and K2 isolated from pus and urine of critically ill patients in tertiary care hospitals in Oman. These strains belong to diverse sequence types (STs), namely ST-23(K1), ST-231(K2), ST-881(K2), and ST-14(K2). To study their virulence, a Galleria mellonella model and resistance to human serum killing were used. The G. mellonella model revealed that the K1/ST-23 isolate was the most virulent, as 50% of the larvae died in the first day, followed by isolate K2/ST-231 and K2/ST-14, for which 75% and 50% of the larvae died in the second day, respectively. Resistance to human serum killing showed there was complete inhibition of bacterial growth of all four isolates by the end of the first hour and up to the third hour. Whole genome sequencing (WGS) revealed that hvKp strains display a unique genetic arrangement of k-loci. Whole-genome single-nucleotide polymorphism-based phylogenetic analysis revealed that these hvKp isolates were phylogenetically distinct, belonging to diverse clades, and belonged to different STs in comparison to global isolates. For ST-23(K1), ST-231(K2), ST-881(K2), and ST-14(K2), there was a gradual decrease in the number of colonies up to the second to third hour, which indicates neutralization of bacterial cells by the serum components. However, this was followed by a sudden increase of bacterial growth, indicating possible resistance of bacteria against human serum bactericidal activity. This is the first report from Oman detailing the WGS of hvKp clinical isolates and assessing their resistance and virulence genomics, which reinforce our understanding of their epidemiology and dissemination in clinical settings.

Comparative Genomic Analysis Reveals the Emergence of ST-231 and ST-395 Klebsiella pneumoniae Strains Associated with the High Transmissibility of blaKPC Plasmids.

Conjugative transposons in Gram-negative bacteria have a significant role in the dissemination of antibiotic-resistance-conferring genes between bacteria. This study aims to genomically characterize plasmids and conjugative transposons carrying integrons in clinical isolates of Klebsiella pneumoniae. The genetic composition of conjugative transposons and phenotypic assessment of 50 multidrug-resistant K. pneumoniae isolates from a tertiary-care hospital (SQUH), Muscat, Oman, were investigated. Horizontal transferability was investigated by filter mating conjugation experiments. Whole-genome sequencing (WGS) was performed to determine the sequence type (ST), acquired resistome, and plasmidome of integron-carrying strains. Class 1 integrons were detected in 96% of isolates and, among integron-positive isolates, 18 stains contained variable regions. Horizontal transferability by conjugation confirmed the successful transfer of integrons between cells and WGS confirmed their presence in conjugative plasmids. Dihydrofolate reductase (dfrA14) was the most prevalent (34.8%) gene cassette in class 1 integrons. MLST analysis detected predominantly ST-231 and ST-395. BlaOXA-232 and blaCTX-M-15 were the most frequently detected carbapenemases and beta-lactamases in the sequenced isolates. This study highlighted the high transmissibility of MDR-conferring conjugative plasmids in clinical isolates of K. pneumoniae. Therefore, the wise use of antibiotics and the adherence to effective infection control measures are necessary to limit the further dissemination of multidrug-resistant bacteria.

Evaluation of six commercial and in-house phenotypic tests for detection of AmpC ß-lactamases: is routine detection possible?

Background: Phenotypic characterization of the prevalent AmpC ß-lactamases in clinical isolates is essential for making informed empirical decisions and critical for strengthening antimicrobial stewardship programmes. This study focused on assessing six assays, two in-house and four commercial phenotypic tests for detection of AmpC, to study the feasibility of making its detection a routine diagnostic microbiology laboratory activity. Methods: A total of 116 non-duplicate Gram-negative bacteria that were resistant to third-generation cephalosporins and amoxicillin/clavulanic acid, and resistant or susceptible to piperacillin/tazobactam and carbapenems, were screened by cefoxitin discs for AmpC. These isolates were subjected to two in-house (AmpC Tris-EDTA and disc approximation) methods and four commercial tests: D69C AmpC Detection Set; D72C ESBL, AmpC & Carbapenemase Detection Set; combination disc test: ESBL + AmpC Screen Disc Kit; and AmpC MIC Test Strip for confirmation of AmpC production. Ten whole-genome-sequenced AmpC-confirmed Gram-negative isolates were used as positive controls and one as a negative control. Results: The prevalence of AmpC ß-lactamases was 16%. Escherichia coli was a major carrier of plasmid-mediated AmpC (26.5%), followed by Klebsiella pneumoniae (23.4%). Phenotypically, 61% of AmpCs were detected by Tris-EDTA (accuracy: 73.8%), 76% by disc approximation (accuracy: 89.2%), 75% by the D69C AmpC Detection Set (accuracy: 95.4%), 74% by the D72C AmpC, ESBL & Carbapenemase Detection Set (accuracy: 95.4%), 76% by the combination disc test (accuracy: 95.4%) and 63% by AmpC MIC Test Strip (accuracy: 87.7%). The sensitivity and specificity of D69C were 97.9% and 88.2%, respectively, and 95.9% and 93.8% for the combination disc test, while for the disc approximation test and D72C they were 93.9% and 75%, and 93.9% and 100%, respectively. Screening by cefoxitin screening was less sensitive (75%) and specific (25%). Disc approximation and the combination disc test detect AmpC in Enterobacterales and also Pseudomonas aeruginosa and Acinetobacter species. Conclusions: We recommend the in-house disc approximation test and the commercial D69C, as well as the combination disc test, as excellent tools for detection of AmpC. The cefoxitin test overcalls AmpC and cannot be considered a good stand-alone test for AmpC detection.

Trends of antimicrobial resistance in patients with complicated urinary tract infection: Suggested empirical therapy and lessons learned from a retrospective observational study in Oman.

Background: Complicated urinary tract infection (cUTI) is defined as an infection associated with structural, functional, or metabolic abnormalities of the genitourinary tract. These infections are caused frequently by multidrug-resistant Gram-negative bacilli. The rapid emergence of extended-spectrum beta-lactamase (ESBL), AmpC, and carbapenemase (CR) producers has made the treatment of such infections increasingly more challenging. Objectives: The aims of the present study were threefold: to assess the clinical profile, trends in etiology, and antimicrobial susceptibility profile in cUTI over the past 10 years at a tertiary care center in Oman as an interrupted time series on the one hand and to develop guidelines for empirical management of such cases on the other. Materials and Methods: We conducted a retrospective analysis of cUTI in patients presenting at Sultan Qaboos University Hospital over 3 years (2008, 2013, and 2018) covering a span of 10 years. Data were obtained from the patient's electronic records in the hospital information system. Analysis was done using the Statistical Package for Social Sciences program (SPSS), version 23. Results: Among the 650 cases of cUTI, 284 (44%) were males and 366 (56%) were females, with dysuria being the most common symptom (34%). The biggest risk factor for developing cUTI was diabetes (35%). The predominant pathogen was Escherichia coli (53%), followed by Klebsiella spp. (16%), Enterococcus faecalis (7%), Pseudomonas aeruginosa (7%), Candida spp. (2%), and Enterobacter cloacae (2%). Over the years, E. coli emerged as the predominant ESBL and AmpC producer, Acinetobacter baumannii as the multidrug-resistant bug, and Klebsiella pneumoniae as the major carbapenem-resistant Enterobacterales (CRE) producer. Nitrofurantoin emerged as the most effective drug for cystitis. Aminoglycosides, piperacillin-tazobactam, and carbapenems demonstrated the highest activity with an overall resistance of less than 10%. Higher resistance (30%) was observed against cephalosporins, fluoroquinolones, and trimethoprim/sulfamethoxazole. Analysis of the 10-year trend threw up some unexpected results. As expected, resistance increased from 2008 to 2013. Surprisingly, however, antimicrobial resistance in 2018 was lower against majority of the antimicrobials compared to 2013. Conclusion: There is a paucity of data for developing evidence-based guidelines management of cUTI. Targeted antibiograms and not cumulative antibiograms are essential for promoting appropriate prescribing and optimizing patient care. The welcome decline in resistance may be attributed cascade reporting, introduction of more ID physicians. Another possibility is increased utilization of fluoroquinolones which spared the other groups of antimicrobials. Judicious heterogeneous mixing of antimicrobials should be spearheaded in both cystitis and pyelonephritis so that there is no undue pressure on one drug. We strongly recommend carbapenem-sparing protocols in treatment of cUTI when anticipating augmented resistance due to AmpC production. Synergistic combinations such as piperacillin-tazobactam plus aminoglycosides/fluoroquinolones may be prescribed. In sepsis, however, carbapenems are the drugs of choice.

Assessment of In-Vitro Synergy of Fosfomycin with Meropenem, Amikacin and Tigecycline in Whole Genome Sequenced Extended and Pan Drug Resistant Klebsiella Pneumoniae: Exploring A Colistin Sparing Protocol.

Fosfomycin has emerged as a very useful antimicrobial in management of extremely drug resistant (XDR) and pan drug resistant (PDR) Klebsiella pneumoniae. In this study, we assessed in-vitro synergy of colistin sparing combinations of fosfomycin (FOS) with meropenem (MEM), tigecycline (TGC) and amikacin (AK) against XDR and PDR Klebsiella pneumoniae. METHOD: Non-replicate fully characterised 18 clinical isolates of K. pneumoniae (15 XDR and 3 PDR strains) were subjected to in-vitro synergy testing by checkerboard and time kill assay. Combinations tested were FOS-MEM, FOS-TGC and FOS-AK with glucose-6-phosphate being incorporated in all runs.WGS was carried out on the Illumina next-generation sequencing platform. RESULTS: FOS-MEM and FOS-AK both demonstrated excellent synergy against all PDRs and all but one XDR. Synergy led to lowering of MICs to susceptible breakpoints. FOS-TGC demonstrated antagonism. MLST-231 K. pneumoniae predominated (14), followed by ST-395 (3) and ST147 (1). Majority harboured OXA-232 (n = 15), while n = 2 carried NDM-1 type and n = 1 co-carried NDM-5 + OXA-232. Mortality was high in both ST-231 (57.1%) and ST-395 (66.6%). Synergy was observed despite widespread presence of resistance markers against aminoglycosides [aph(3')-Ic, aacA4, and rmtf], beta-lactams [blaSHV-11, blaTEM-1b, blaCTX-M-15, and blaOXA-232], fosfomycin [fosA6 and fosA5] and presence of porin proteins OmpK37, OmpA and K. pneumoniae antibiotic efflux pumps Kpn F, H, G, and E. CONCLUSION: FOS + MEM and FOS + AK are excellent colistin sparing combinations against ST 231, ST-395 and ST-147 XDR and PDR K. pneumoniae. FOS with fewer side effects than colistin, excellent tissue distribution and minimal side effects may be recommended in combination with meropenem.

Effects of Bacterial Metabolites on the Immune System: Enemies and Friends.

Metabolites produced by bacteria can influence the immune system. These metabolites are produced by pathogenic bacteria as well as the friendly microbiota. This review sheds light on the major bacterial metabolites and their structures. It also describes the capacity of these molecules to stimulate and inhibit the immune responses in a way that affects their capacity to control different diseases.

Proteomics: Concepts and applications in human medicine.

Proteomics is the complete evaluation of the function and structure of proteins to understand an organism's nature. Mass spectrometry is an essential tool that is used for profiling proteins in the cell. However, biomarker discovery remains the major challenge of proteomics because of their complexity and dynamicity. Therefore, combining the proteomics approach with genomics and bioinformatics will provide an understanding of the information of biological systems and their disease alteration. However, most studies have investigated a small part of the proteins in the blood. This review highlights the types of proteomics, the available proteomic techniques, and their applications in different research fields.

Whole Genome Sequencing of Methicillin-Resistant Staphylococcus epidermidis Clinical Isolates Reveals Variable Composite SCCmec ACME among Different STs in a Tertiary Care Hospital in Oman.

Staphylococcus epidermidis has been recently recognized as an emerging nosocomial pathogen. There are concerns over the increasing virulence potential of this commensal due to the capabilities of transferring mobile genetic elements to Staphylococcus aureus through staphylococcal chromosomal cassette (SCCmec) and the closely related arginine catabolic mobile element (ACME) and the copper and mercury resistance island (COMER). The potential pathogenicity of S. epidermidis, particularly from blood stream infections, has been poorly investigated. In this study, 24 S. epidermidis isolated from blood stream infections from Oman were investigated using whole genome sequence analysis. Core genome phylogenetic trees revealed one third of the isolates belong to the multidrug resistance ST-2. Genomic analysis unraveled a common occurrence of SCCmec type IV and ACME element predominantly type I arranged in a composite island. The genetic composition of ACME was highly variable among isolates of same or different STs. The COMER-like island was absent in all of our isolates. Reduced copper susceptibility was observed among isolates of ST-2 and ACME type I, followed by ACME type V. In conclusion, in this work, we identify a prevalent occurrence of highly variable ACME elements in different hospital STs of S. epidermidis in Oman, thus strongly suggesting the hypothesis that ACME types evolved from closely related STs.

Reliability of carbapenem inactivation method (CIM) and modified carbapenem inactivation method (mCIM) for detection of OXA-48-like and NDM-1.

PURPOSE: Carbapenem inactivation method (CIM) and modified carbapenem inactivation method (mCIM) were recently developed for rapid detection of carbapenemase producing Gram negative bacilli (CP-GNB). In this study we compared the ability of modified Hodge test (MHT), CIM and mCIM to identify CP-GNB in Oman and India. METHODS: Fifty fully characterized and genotyped CP-GNB (26 OXA-48-like, 2 NDM-1 from Oman and 22 NDM-1 from India) and 8 AmpC as controls in India were subjected to MHT, CIM, mCIM and mCIM with in-house modifications. Wilcoxon paired test and receiver operating characteristics (ROC) were utilised for statistical analysis. RESULTS: Isolates were predominantly OXA-48-like genes producing Klebsiella pneumoniae from Oman and NDM-1 producing Escherichia coli from India. MHT was positive in all except one OXA-48-like producers and in 70.8 â€‹% of the NDM-1 isolates. The sensitivity of CIM in detecting 0XA-48 like and NDM-1 carbapenemases were 39.2% and 87.5% respectively. mCIM at 4 â€‹h detected 92.3 â€‹% and 79.1% of 0XA-48 and NDM-1 respectively. Using receiver operative characteristics (ROC), highest sensitivity and specificity for detection of OXA-48-like was obtained by mCIM at 4 â€‹h at cut off 17 â€‹mm while for NDM-1 CIM was the test of choice at 16 â€‹mm. CONCLUSION: CIM and mCIM are simple, cheap and easy tests to perform. CIM gave excellent results with NDM1 strains while it was quite poor in predicting OXA-48-like. We recommend CIM and eCIM for rapid identification of NDM-1 producers and mCIM at 4 â€‹h and MHT for detection of OXA-48-like. No one method can correctly detect both genotypes. As determined by ROC curves a zone of inhibition of 17 â€‹mm was considered adequate for detection of OXA-48-like and 16 â€‹mm of NDM-1 by mCIM at 4 â€‹h and CIM respectively.

Integrase-Controlled Excision of Metal-Resistance Genomic Islands in Acinetobacter baumannii.

Genomic islands (GIs) are discrete gene clusters encoding for a variety of functions including antibiotic and heavy metal resistance, some of which are tightly associated to lineages of the core genome phylogenetic tree. We have investigated the functions of two distinct integrase genes in the mobilization of two metal resistant GIs, G08 and G62, of Acinetobacter baumannii. Real-time PCR demonstrated integrase-dependent GI excision, utilizing isopropyl ß-d-1-thiogalactopyranoside IPTG-inducible integrase genes in plasmid-based mini-GIs in Escherichia coli. In A. baumannii, integrase-dependent excision of the original chromosomal GIs could be observed after mitomycin C induction. In both E. coli plasmids and A. baumannii chromosome, the rate of excision and circularization was found to be dependent on the expression level of the integrases. Susceptibility testing in A. baumannii strain ATCC 17978, A424, and their respective ΔG62 and ΔG08 mutants confirmed the contribution of the GI-encoded efflux transporters to heavy metal decreased susceptibility. In summary, the data evidenced the functionality of two integrases in the excision and circularization of the two Acinetobacter heavy-metal resistance GIs, G08 and G62, in E. coli, as well as when chromosomally located in their natural host. These recombination events occur at different frequencies resulting in genome plasticity and may participate in the spread of resistance determinants in A. baumannii.

Lipopeptidomimetics derived from teixobactin have potent antibacterial activity against Staphylococcus aureus.

A series of lipopeptidomimetics derived from teixobactin have been prepared that probe the role of residues (1-6) as a membrane anchor and the function of enduracididine. The most active compounds, with a farnesyl tail and End10 to Lys10 or Orn10 substitution have potent activity (MIC 8 µg mL-1) against S. aureus. These results pave the way for the synthesis of simple, cost-effective yet potent lipopeptidomimetic antimicrobials.

Promysalin is a salicylate-containing antimicrobial with a cell-membrane-disrupting mechanism of action on Gram-positive bacteria.

Promysalin was previously described as a narrow spectrum molecule with a unique species-specific activity against Pseudomonas aeruginosa. Here we demonstrate that promysalin is active against Gram-positive and Gram-negative bacteria using a microdilution assay. Promysalin acts on Gram-positive bacteria with a mechanism of action involving cell membrane damage with leakage of intracellular components. The evaluation of MICs and MBCs on 11 promysalin analogs, synthesized utilizing diverted total synthesis, allowed the identification of the structural moieties potentially involved in cell membrane interaction and damage. The mechanism of action of promysalin against Gram-negative bacteria is still not clarified, even if a synergistic effect with the bisguanidine chlorhexidine on cell membrane disruption has been observed.

Dissemination of Novel Antimicrobial Resistance Mechanisms through the Insertion Sequence Mediated Spread of Metabolic Genes.

The widely used biocide triclosan selectively targets FabI, the NADH-dependent trans-2-enoyl-acyl carrier protein (ACP) reductase, which is also an important target for the development of narrow spectrum antibiotics. The analysis of triclosan resistant Staphylococcus aureus isolates had previously shown that in about half of the strains, the mechanism of triclosan resistance consists on the heterologous duplication of the triclosan target gene due to the acquisition of an additional fabI allele derived from Staphylococcus haemolyticus (sh-fabI). In the current work, the genomic sequencing of 10 of these strains allowed the characterization of two novel composite transposons TnSha1 and TnSha2 involved in the spread of sh-fabI. TnSha1 harbors one copy of IS1272, whereas TnSha2 is a 11.7 kb plasmid carrying TnSha1 present either as plasmid or in an integrated form generally flanked by two IS1272 elements. The target and mechanism of integration for IS1272 and TnSha1 are novel and include targeting of DNA secondary structures, generation of blunt-end deletions of the stem-loop and absence of target duplication. Database analyses showed widespread occurrence of these two elements in chromosomes and plasmids, with TnSha1 mainly in S. aureus and with TnSha2 mainly in S. haemolyticus and S. epidermidis. The acquisition of resistance by means of an insertion sequence-based mobilization and consequent duplication of drug-target metabolic genes, as observed here for sh-fabI, is highly reminiscent of the situation with the ileS2 gene conferring mupirocin resistance, and the dfrA and dfrG genes conferring trimethoprim resistance both of which are mobilized by IS257. These three examples, which show similar mechanisms and levels of spread of metabolic genes linked to IS elements, highlight the importance of this genetic strategy for recruitment and rapid distribution of novel resistance mechanisms in staphylococci.

Susceptibility to chlorhexidine amongst multidrug-resistant clinical isolates of Staphylococcus epidermidis from bloodstream infections.

The emergence of Staphylococcus isolates with reduced susceptibility to chlorhexidine is being increasingly reported. We present an update to a previous report showing the continuing efficacy of chlorhexidine-based infection control measures against Staphylococcus aureus over 6 years. In this study, qacA/B genes were screened in Staphylococcus isolates collected over another 6 years in the same intensive care unit in Scotland where chlorhexidine baths form an essential component of long-term control of nosocomial infections. Consistent with our previous study, we report minimal presence of qacA/B in S. aureus strains from screening samples and bacteraemia patients but the new finding of a high proportion of qacA/B carriage in Staphylococcus epidermidis associated with reduced susceptibility to chlorhexidine. S. epidermidis isolates positive for qacA/B were clonally diverse, although 65% of isolates belonged to the multidrug-resistant (MDR) clone ST2. These findings raise concerns in relation to the selection of MDR strains by chlorhexidine and are important in the context of recent evidence emphasising the benefits of targeting bloodstream infections associated with coagulase-negative staphylococci.