Staphylococcus aureus resistance to non-β-lactam antibiotics: global genomic epidemiology and machine learning feasibility assessment.

PurposeSeveral subtypes of plasmid-mediated fosfomycin resistance gene fosA in Enterobacteriaceae have been reported worldwide and have caused concern. The present study characterized a novel member of fosA gene located on a plasmid from Escherichia coli.Materials and MethodsA fosfomycin-resistant E. coli isolate PK9 was recovered from a chicken meat sample in 2018. The presence of fosA genes was detected by PCR and sequencing. Whole-genome sequencing (WGS), conjugation, and cloning were performed to identify the mechanism responsible for fosfomycin resistance. Oxford Nanopore MinION sequencing was carried out to characterize the plasmid carrying fosfomycin resistance gene and the genetic context of the novel fosA variant.ResultsA novel fosA gene with significant homology (>98%) with fosA6 and fosA5 genes was identified by WGS and was named fosA10. FosA10 shared 56.1% to 98.6% amino acid sequence identity with other reported plasmid-mediated FosA enzymes. Fosfomycin resistance and fosA10 gene were successfully transferred to E. coli C600 by conjugation. Cloning confirmed that FosA10 could confer fosfomycin resistance (MIC > 128 μg/mL). The fosA10 gene was localized on a 53kb IncFII (F35:A-:B-) plasmid. The ∆lysR-fosA10-∆hp fragment (4328 bp), located between two copies of IS10R, showed 100% identity with the chromosomal sequences of 17 Klebsiella pneumoniae strains of ST664 and one of ST3821 in GenBank.ConclusionOur findings indicated that the fosA10 gene of E. coli might be captured from the chromosome of K. pneumoniae by IS10, which further demonstrated that K. pneumoniae might act as a reservoir of fosA-like genes acquired by E. coli.

The identification of genes associated with resistance has the potential to facilitate the development of novel diagnostic tests and treatment methods. The objective of this study was to examine the antibiotic resistance and Fosfomycin resistance genes in uropathogenic Escherichia coli (UPEC) in patients in Baghdad, Iraq. After analyzing 250 urine samples using various identification methods, including the examination of morphological characteristics, biochemical tests, and genetic detection, it was determined that E. coli was the most common bacteria present, accounting for 63.6% of the samples. Antibiotic susceptibility testing showed a significant prevalence of resistance to various antibiotics, with 99.3% of E. coli isolates exhibiting multiple drug resistance (MDR). Fosfomycin showed antibacterial properties against UPEC. The minimum inhibitory concentration (MIC) ranged from 512 to 1024μg/mL, while the minimum bactericidal concentration (MBC) was 2048μg/mL. In the time-kill assay, fosfomycin was effective against fosfomycin-resistant isolates within 8-12h. The genetic determinants associated with fosfomycin resistance were examined through the utilization of polymerase chain reaction (PCR). The findings indicated that the genes murA, glpT, and cyaA were detected in all the isolates when genomic DNA was used as a template. However, all the tests yielded negative results when plasmid was used as a template. The genes fosA3 and fosA4 were detected in 8.6% and 5% of the isolates when genomic DNA was used as a template. When plasmid was used as a template, the genes fosA3 and fosA4 were found in 5.7% and 2.9% of the isolates, respectively. In conclusion, there is an increasing problem with antibiotic resistance in UPEC, with elevated rates of resistance to several antibiotics. The study also offers novel insights into the genetic foundation of fosfomycin resistance in UPEC.

The revival of fosfomycin

M. morganii is a species of the genus Morganella in the family Enterobacteriaceae. This species primarily causes infections of postoperative wounds and the urinary tract. Some isolates of M. morganii exhibit resistance to multiple antibiotics due to multidrug resistance traits, complicating clinical treatment; thus, there is a growing need to elucidate the resistance mechanisms of this pathogen. A total of 658 bacterial strains were isolated from anal fecal swabs from poultry and livestock and from the surrounding environment in Wenzhou, China, via plate streaking. The full genome sequences of the bacteria were obtained via next-generation sequencing platforms. The standard agar dilution method was employed to determine the minimum inhibitory concentrations (MICs) of various antimicrobial agents. The resistance gene (fosA13) of the isolate was identified using the Comprehensive Antibiotic Resistance Database (CARD) and confirmed via molecular cloning. The FosA13 protein encoded by the novel resistance gene fosA13 was expressed with the vector pCold I, and its enzyme kinetics parameters were characterized. The genetic background and evolutionary process of the sequence of this novel resistance gene were analyzed by means of bioinformatics methods. In this study, we identified a new chromosomally encoded fosfomycin resistance gene, designated fosA13, from the M. morganii isolate DW0548, which was isolated from poultry on a farm in Wenzhou, China. Compared with the control strain (pUCP19/DH5α), the recombinant strain carrying fosA13 (pUCP19-fosA13/DH5α) presented a fourfold increase in the MIC value for fosfomycin. The enzyme kinetics data of FosA13 revealed effective inactivation of fosfomycin, with a k cat /K m of (1.50 ± 0.02)×104 M-1·s-1. Among functionally characterized resistance proteins, FosA13 presented the highest amino acid (aa) homology (55.6%) with FosA. FosA13 also contained essential functional residues of FosA proteins. The isolate M. morganii DW0548 presented high MIC values (≥ 8 μg/mL) for 5 classes of antimicrobials, namely, aminoglycosides, β-lactams, quinolones, tetracycline, and chloramphenicol, but only two functionally characteristic antimicrobial resistance genes (ARGs) have been identified in the complete genome: a β-lactam resistance gene (bla DHA-16) and a phenol resistance gene (catII). These findings indicate that in addition to the novel resistance gene identified in this work, other uncharacterized resistance mechanisms might exist in M. morganii DW0548. A novel chromosomal fosfomycin resistance gene, fosA13, was identified in an animal M. morganii isolate, and its enzymatic parameters were characterized. This protein shares the highest aa identity of 55.6% with the functionally characterized protein FosA and has all the essential functional residues of FosA proteins. Exploring more antimicrobial resistance mechanisms of this pathogen would help clinicians choose effective drugs to treat infectious diseases in animal husbandry and clinical practice and facilitate the development of methods to prevent the spread of resistance between bacteria of different species.

Fosfomycin has gained attention as a combination therapy for methicillin-resistant Staphylococcus aureus infections. Hence, the detection of novel fosfomycin-resistance mechanisms in S. aureus is important. Here, the minimal inhibitory concentrations (MICs) of fosfomycin in CC1 methicillin-resistant S. aureus were determined. The pangenome analysis and comparative genomics were used to analyse CC1 MRSA. The gene function was confirmed by cloning the gene into pTXΔ. A phylogenetic tree was constructed to determine the clustering of the CC1 strains of S. aureus. We identified a novel gene, designated fosY, that confers fosfomycin resistance in S. aureus. The FosY protein is a putative bacillithiol transferase enzyme sharing 65.9–77.5% amino acid identity with FosB and FosD, respectively. The function of fosY in decreasing fosfomycin susceptibility was confirmed by cloning it into pTXΔ. The pTX-fosY transformant exhibited a 16-fold increase in fosfomycin MIC. The bioinformatic analysis showed that fosY is in a novel genomic island designated RI fosY (for “resistance island carrying fosY”) that originated from other species. The global phylogenetic tree of ST1 MRSA displayed this fosY-positive ST1 clone, originating from different regions, in the same clade. The novel resistance gene in the fos family, fosY, and a genomic island, RI fosY , can promote cross-species gene transfer and confer resistance to CC1 MRSA causing the failure of clinical treatment. This emphasises the importance of genetic surveillance of resistance genes among MRSA isolates.

BackgroundThe temporal and longitudinal trends of β-lactamases and their associated susceptibility patterns were analyzed for Escherichia coli and Klebsiella pneumoniae isolates consecutively collected in 56 United States hospitals during 2016–2020.MethodsIsolates (n = 19 453) were susceptibility tested by reference broth microdilution methods. Isolates that displayed minimum inhibitory concentration (MIC) values ≥2 mg/L for at least 2 of the following compounds—ceftazidime, ceftriaxone, aztreonam, or cefepime—or resistance to the carbapenems were submitted to whole genome sequencing for identification of β-lactamases. Longitudinal and temporal trends were determined by slope coefficient. New CTX-M and OXA-1 variants were characterized.ResultsExtended-spectrum β-lactamases (ESBLs) were detected among 88.0% of the isolates that displayed elevated cephalosporin/aztreonam MICs without carbapenem resistance. blaCTX-M-15 was detected among 55.5% of the ESBL producers. ESBL rates were stable over time, but significant increases were noted among bloodstream infection and K pneumoniae isolates, mainly driven by an increase in blaCTX-M. Carbapenem resistance and carbapenemase genes were noted among 166 and 145 isolates, respectively, including 137 blaKPC, 6 blaSME, 3 blaOXA-48–like, and 3 blaNDM. Ceftazidime-avibactam and carbapenems were very active (>99% susceptibility) against ESBL producers without carbapenem resistance. Ceftazidime-avibactam inhibited 97.0% of the carbapenem-resistant isolates. This agent and meropenem-vaborbactam inhibited 96.4% and 85.0% of the 2020 isolates, respectively.ConclusionsOverall, ESBL-producing isolates were stable, but an increase was noted for K pneumoniae isolates driven by CTX-M production. Carbapenem-resistant Enterobacterales rates decreased in the study period. The prevalence of metallo-β-lactamases and OXA-48–like remains low. Continuous surveillance of β-lactamase–producing isolates is prudent.

With the spread of multi-drug-resistant (MDR) bacteria and the lack of effective antibiotics to treat them, developing new therapeutic methods and strategies is essential. In this study, we evaluated the antibacterial and antibiofilm activity of different formulations composed of ibuprofen (IBP), acetylsalicylic acid (ASA), and dexamethasone sodium phosphate (DXP) in combination with ciprofloxacin (CIP), gentamicin (GEN), cefepime (FEP), imipenem (IPM), and meropenem (MEM) on clinical isolates of Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa) as well as the transcription levels of biofilm-associated genes in the presence of sub-MICs of IBP, ASA, and DXP. The minimal inhibitory concentrations (MICs), minimal biofilm inhibitory concentrations (MBICs), and minimum biofilm eradication concentrations (MBECs) of CIP, GEN, FEP, IPM, and MEM with/without sub-MICs of IBP (200 µg/mL), ASA (200 µg/mL), and DXP (500 µg/mL) for the clinical isolates were determined by the microbroth dilution method. Quantitative real-time-PCR (qPCR) was used to determine the expression levels of biofilm-related genes, including icaA in S. aureus and algD in P. aeruginosa at sub-MICs of IBP, ASA, and DXP. All S. aureus isolates were methicillin-resistant S. aureus (MRSA), and all P. aeruginosa were resistant to carbapenems. IBP decreased the levels of MIC, MBIC, and MBEC for all antibiotic agents in both clinical isolates, except for FEP among P. aeruginosa isolates. In MRSA isolates, ASA decreased the MICs of GEN, FEP, and IPM and the MBICs of IPM and MEM. In P. aeruginosa, ASA decreased the MICs of FEP, IPM, and MEM, the MBICs of FEP and MEM, and the MBEC of FEP. DXP increased the MICs of CIP, GEN, and FEP, and the MBICs of CIP, GEN, and FEP among both clinical isolates. The MBECs of CIP and FEP for MRSA isolates and the MBECs of CIP, GEN, and MEM among P. aeruginosa isolates increased in the presence of DXP. IBP and ASA at 200 µg/mL significantly decreased the transcription level of algD in P. aeruginosa, and IBP significantly decreased the transcription level of icaA in S. aureus. DXP at 500 µg/mL significantly increased the expression levels of algD and icaA genes in S. aureus and P. aeruginosa isolates, respectively. Our findings showed that the formulations containing ASA and IBP have significant effects on decreasing the MIC, MBIC, and MBEC levels of some antibiotics and can down-regulate the expression of biofilm-related genes such as icaA and algD. Therefore, NSAIDs represent appropriate candidates for the design of new antibacterial and antibiofilm therapeutic formulations.

Objective To test the susceptibility of Penicilliosis marneffei (PM) isolates from Guangxi bamboo rats and patients to voriconazole and several commonly used antifungal agents.Methods According to the Clinical and Laboratory Standards Institute (CLSI) M27-A2 and M38-A document,a microdilution method was used to determine the minimum inhibitory concentration (MIC) of voriconazole,itraconazole,terbinafine,amphotericin B,and fluconazole against mycelial phase (25 ℃) and yeast phase (37 ℃) of 14 PM isolates from Guangxi Bamboo rats and 25 PM isolates from patients.The difference in MIC of the antifungals was assessed by two-sample t test between Bamboo rat PM isolates and clinical PM isolates,and by paired t test between the mycelial and yeast phase of PM isolates.Results The MIC ranges of voriconazole,itraconazole,terbinafine,amphoteriein B and fluconazole were 0.0313-0.1250,0.1250-1.0000,0.0313-0.5000,0.2500-4.0000,2.0000-8.0000 mg/L,respectively for mycelial phase of Bamboo rat PM isolates,0.0078-0.2500,0.0313-0.5000,0.0313-1.0000,0.2500-2.0000,1.0000-8.0000 mg/L,respectively for yeast phase of Bamboo rat PM isolates,0.0313-0.2500,0.0625-1.0000,0.0313-1.0000,0.2500-4.0000,2.0000-32.0000 mg/L,respectively for mycelial phase of clinical PM isolates,0.0039-0.2500,0.0313-0.5000,0.0313-2.0000,0.1250-2.0000,2.0000-16.0000 mg/L,respectively for yeast phase of clinical PM isolates.None of the PM isolates was resistant to any of the antifungals.The MIC of voriconazole was found to be the lowest for PM isolates from both Bamboo rats and patients at the same temperature (37 ℃ or 25 ℃),followed by itraconazole,terbinafine,amphotericin B and fluconazole.Statistical difference was found in the MIC values of itraconazole,terbinafine,amphotericin B between the yeast and mycelial phase of the same PM isolate,but not found in antifungal MIC values between Bamboo rat isolates and clinical isolates at the same phase.Conclusions Of the tested drugs,voriconazole shows the strongest antifungal potency. The PM isolates from Guangxi Bamboo rats are similar to clinical PM isolates in the sensitivity to voriconazole,itraconazole,terbinafine,amphotericin B and fluconazole.The phase of PM isolates may affect their susceptibility to itraconazole,amphotericin B and terbinafine. Key words: Penicillium; Antifungal agents; Microbial sensitivity tests

Background: Many clinical isolates of Pseudomonas aeruginosa exhibit antibiotic-resistance and it is the main cause of hospital infections. Objectives: The current study aimed at evaluating the diversity of genes coding for antibiotic resistance in clinical and environmental isolates of P. aeruginosa in Hamadan, West of Iran. Methods: In the current cross sectional study, 100 clinical and 50 environmental isolates of P. aeruginosa were collected from October 2013 to May 2014. The standard disk diffusion method (SDD) was performed to test antibiotic susceptibility. All isolates were evaluated by the polymerase chain reaction (PCR) for the presence of class 1 integrons and genes encoding resistance to aminoglycoside antibiotics. The relationship between antibiotic resistance and integrons was tested by the 2-tailed chi-square (χ 2 ) or the Fisher exact tests. P values ≤ 0.05 were considered statistically significant. Results: The most common antibiotic resistance was to cotrimoxazole (61.3%). The least observed resistance was to meropenem (9.3%) and piperacillin /tazobactam (12%). The result of minimum inhibitory concentration (MIC) for amikacin showed that 20% of the strains were resistant, 15% had intermediate resistance, and 65% were sensitive. Class 1 integrons were found in 57% of the isolates. The aminoglycoside-resistance cassette included aadA (confers resistance to streptomycin and spectinomycin) and aphA1 (encoded aminoglycoside 3’-phosphotransferase) genes were the most prevalent in the isolates. Conclusions: Antibiotic resistance had a high prevalence in clinical isolates of P. aeruginosa and was commonly associated with class 1 integrons. The knowledge of drug resistance patterns helps to apply effective antibiotic treatments and appropriate infection control measures to prevent the spread of infection in hospitals.

Rapid bactericidal and anti-virulence effects of Quercus infectoria gall extract against the pharyngitis causing pathogen Streptococcus pyogenes.

MurA [UDP-N-acetylglucosamine (UDP-NAG) enolpyruvyl transferase] is a key enzyme involved in bacterial cell wall peptidoglycan synthesis and a target for the antimicrobial agent fosfomycin, a structural analog of the MurA substrate phosphoenol pyruvate. In this study, we identified, cloned and sequenced a novel murA gene from an environmental isolate of Vibrio fischeri that is naturally resistant to fosfomycin. The fosfomycin resistance gene was isolated from a genomic DNA library of V. fischeri. An antimicrobial agent hypersensitive strain of Escherichia coli harboring murA from V. fischeri exhibited a high fosfomycin resistance phenotype, with minimum inhibitory concentration of 3,000 microg/ml. The cloned murA gene was 1,269 bp long encoding a 422 amino acid polypeptide with an estimated pI of 5.0. The deduced amino acid sequence of the putative protein was identified as UDP-NAG enolpyruvyl transferase by homology comparison. The MurA protein with an estimated molecular weight of 44.7 kDa was expressed in E. coli and purified by affinity chromatography. MurA of V. fischeri will be a useful target to identify potential inhibitors of fosfomycin resistance in pharmacological studies.

Background/aimFosfomycin has regained importance owing to its unique mechanism of action and effectiveness against extended-spectrum β-lactamase-producing Gram-negative bacteria. This study aimed to evaluate the biological fitness cost associated with fosfomycin resistance and its impact on biofilm formation in clinical Enterobacteriaceae isolates.Materials and methodsA total of 78 Escherichia coli and 34 Klebsiella pneumoniae strains isolated from urine samples at Ege University Hospital were analyzed. Fosfomycin minimum inhibitory concentrations (MICs) were determined using the reference agar dilution method. Resistance was induced by exposing two K. pneumoniae strains with a fosfomycin MIC of 4 μg/mL and two E. coli strains susceptible to fosfomycin (MIC ≤ 8 μg/mL) to gradually increasing concentrations of the antibiotic. Biofilm-forming capacities, growth rates, and expression levels of selected virulence genes (fimH and papC in E. coli; entB, mrkD, uge, wabG, and ycfM in K. pneumoniae) were compared between variants with low and high fosfomycin MICs.ResultsOf the 78 E. coli isolates, 13 (16.6%) were resistant to fosfomycin. Additionally, eight (23.5%) of 34 K. pneumoniae isolates exhibited high fosfomycin MICs (MIC > 32 μg/mL). No significant differences in biofilm formation were observed between the variants. However, the expression of the fimH gene decreased in one E. coli resistant variant compared with its susceptible counterpart. While the expression of the uge gene decreased in one K. pneumoniae isolate with a high MIC, the expression of the wabG gene increased. Slower growth rates were observed in two fosfomycin-resistant E. coli strains and one K. pneumoniae strain with a high fosfomycin MIC than in their counterparts.ConclusionThese findings suggest that, in the examined isolates, decreased susceptibility to fosfomycin was associated with slower growth, whereas biofilm formation ability remained largely unaffected. Continued surveillance of fosfomycin resistance is essential owing to its potential implications for bacterial fitness and pathogenicity.

Objective. To determine in vitro activity of thiamphenicol and other clinically available antimicrobials against clinical isolates of Haemophilus influenzae, Streptococcus pneumoniae and Streptococcus pyogenes. Materials and Methods. We included in the study 875 clinical isolates from 20 Russian cities during 2018–2019. Among tested strains, 126 were H. influenzae, 389 – S. pneumoniae, 360 – S. pyogenes. Antimicrobial susceptibility testing was performed using broth microdilution method according to ISO 20776-1:2006. AST results were interpreted according to EUCAST v.11.0 clinical breakpoints. Results. The minimum inhibitory concentrations (MICs) of thiamphenicol did not exceed 2 mg/L for 94.4% of H. influenzae strains (MIC50 and MIC90 were 0.5 and 1 mg/L, respectively). Thiamphenicol was active against 76.9% of ampicillin-resistant H. influenzae strains (MIC of thiamphenicol < 2 mg/L). The MIC of thiamphenicol was in the range of 0.06–2 mg/L for 96.7% of S. pneumoniae strains (MIC50 and MIC90 were 0.5 and 2 mg/L, respectively). The MIC of thiamphenicol for 90.6% of S. pneumoniae strains with reduced susceptibility to penicillin (MIC of penicillin > 0.06 mg/L) did not exceed 2 mg/L. A total of 88.1% of S. pneumoniae strains resistant to erythromycin were highly susceptible to thiamphenicol (MIC < 2 mg/L). The MIC of thiamphenicol did not exceed 8 mg/L for 96.1% of S. pyogenes strains (MIC50 and MIC90 were 2 and 4 mg/L, respectively). Conclusions. Thiamphenicol was characterized by relatively high in vitro activity, comparable to that of chloramphenicol, against tested strains of H. influenzae, S. pneumoniae and S. pyogenes, including S. pneumoniae isolates with reduced susceptibility to penicillin.

Phenotypic and WGS-derived antimicrobial resistance profiles of clinical and non-clinical Acinetobacter baumannii isolates from Germany and Vietnam

Prevalence and mechanisms of fosfomycin resistance among KPC-producing Klebsiella pneumoniae clinical isolates in China