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233600.9700Distribution of disinfectant resistant genes in mcr-1-carrying Escherichia coli isolated from children in southern China. BACKGROUND: Colistin, a polymyxin antibiotic, serves as a crucial defense against multidrug-resistant gram-negative bacteria, despite its nephrotoxicity. However, the plasmid-mediated mobilization of the polymyxin resistance gene, mcr-1, presents a significant public health threat. The widespread use of disinfectants has resulted in Escherichia coli (E. coli) carrying mcr-1 also showing disinfectant resistance. The aim of this study is to investigate the distribution of disinfectant genes and resistance to disinfectants in mcr-1-carring E coli from children in the South China. METHODS: We evaluated the distribution of twelve disinfectant-resistance genes by PCR. Evaluated the correlation between disinfectant-resistance genes and resistance to disinfectants and antibiotics. We also examined the correlation between the strains' biofilm formation and the presence of disinfectant-resistance genes. Bioinformatic tools were employed to analyze resistance genes, virulence genes, and insertion sequences. Five strains were randomly selected to examine the effects of sub-inhibitory concentration (sub-MIC) of 8 disinfectants on the expression of the mcr-1 gene by qRT-PCR. RESULTS: The most prevalent of the nine biocide resistance genes were mdfA, sugE(c), ydgE, and ydgF (n = 21; all 100 %). The qacG, qacF, sugE(p) and tehA gene was not detected. Furthermore, benzalkonium chloride (BC) and potassium hydrogen persulfate (PMPS)-based disinfectants were effective against all mcr-1-carrying E. coli strains. The majority of mcr-1 were distributed among the InHI2 plasmid types, although three strains lacked mcr-1 on their plasmids. Biofilm formation was observed in 48 % of the strains. emrD and sitABCD showed significant associations with the susceptibility of the strains to 84 disinfectants (P of 0.0351 and 0.0300). In addition, sitABCD was significantly associated with susceptibility to povidone-iodine (PVP-I) (P value of 0.0062). Compared to the untreated group, stimulation with sub-MIC of peracetic acid (PAA) and PVP-I resulted in decreased or increased mcr-1 expression in five E. coli strains, respectively (P of 0.0011 for PAA and P of 0.0476 for PVP-I). CONCLUSION: BC and PMPS based disinfectants were effective against all mcr-1 carrying E. coli strains. Most of the mcr-1 genes were distributed among the InHI2 plasmid types. The emrD and sitABCD genes are highly associated with resistance to 84 disinfectants, and the sitABCD gene was highly associated with resistance to PVP-I. PVP-I selective pressure may encourage the maintenance of mcr-1 gene in E. coli.202539551109
140610.9694Multicentre study of the burden of multidrug-resistant bacteria in the aetiology of infected diabetic foot ulcers. BACKGROUND: Infected diabetic foot ulcer (IDFU) is a public health issue and the leading cause of non-traumatic limb amputation. Very few published data on IDFU exist in most West African countries. OBJECTIVE: The study investigated the aetiology and antibacterial drug resistance burden of IDFU in tertiary hospitals in Osun state, Nigeria, between July 2016 and April 2017. METHODS: Isolates were cultured from tissue biopsies or aspirates collected from patients with IDFU. Bacterial identification, antibiotic susceptibility testing and phenotypic detection of extended-spectrum beta-lactamase and carbapenemase production were done by established protocols. Specific resistance genes were detected by polymerase chain reaction. RESULTS: There were 218 microorganisms isolated from 93 IDFUs, comprising 129 (59.2%) Gram-negative bacilli (GNB), 59 (27.1%) Gram-positive cocci and 29 (13.3%) anaerobic bacteria. The top five facultative anaerobic bacteria isolated were: Staphylococcus aureus (34; 15.6%), Escherichia coli (23; 10.6%), Pseudomonas aeruginosa (20; 9.2%), Klebsiella pneumoniae (19; 8.7%) and Citrobacter spp. (19; 8.7%). The most common anaerobes were Bacteroides spp. (7; 3.2%) and Peptostreptococcus anaerobius (6; 2.8%). Seventy-four IDFUs (80%) were infected by multidrug-resistant bacteria, predominantly methicillin-resistant S. aureus and GNB producing extended-spectrum β-lactamases, mainly of the CTX-M variety. Only 4 (3.1%) GNB produced carbapenemases encoded predominantly by bla (VIM). Factors associated with presence of multidrug-resistant bacteria were peripheral neuropathy (adjusted odds ratio [AOR] = 4.05, p = 0.04) and duration of foot infection of more than 1 month (AOR = 7.63, p = 0.02). CONCLUSION: Multidrug-resistant facultative anaerobic bacteria are overrepresented as agents of IDFU. A relatively low proportion of the aetiological agents were anaerobic bacteria.202133824857
140020.9693Comparative genomic analysis of Escherichia coli strains obtained from continuous imipenem stress evolution. The carbapenem-resistant Escherichia coli has aroused increasing attention worldwide, especially in terms of imipenem (IMP) resistance. The molecular mechanism of IMP resistance remains unclear. This study aimed to explore the resistance mechanisms of IMP in E. coli. Susceptible Sx181-0-1 strain was induced into resistance strains by adaptive laboratory evolution. The drug resistance spectrum was measured using the disk diffusion and microbroth dilution methods. Whole-genome sequencing and resequencing were used to analyze the nonsynonymous single-nucleotide polymorphisms (nsSNPs) between the primary susceptible strain and resistant strains. The expression levels of these genes with nsSNPs were identified by real-time quantitative PCR (RT-qPCR). Resistance phenotype appeared in the induced 15th generation (induction time = 183 h). Sx181-32 and Sx181-256, which had the minimum inhibitory concentrations of IMP of 8 and 64 µg ml-1, were isolated during continuous subculture exposed to increasing concentrations of IMP, respectively. A total of 19 nsSNPs were observed both in Sx181-32 and Sx181-256, distributed in rpsU, sdaC, zwf, ttuC, araJ, dacC, mrdA, secF, dacD, lpxD, mrcB, ftsI, envZ, and two unknown function genes (orf01892 and orf01933). Among these 15 genes, five genes (dacC, mrdA, lpxD, mrcB, and ftsI) were mainly involved in cell wall synthesis. The mrdA (V338A, L378P, and M574I) and mrcB (P784L, A736V, and T708A) had three amino acid substitutions, respectively. The expression levels of rpsU, ttuC, and orf01933 were elevated in both Sx181-32 and Sx181-256 compared to Sx181-0-1. The expression levels of these genes were elevated in Sx181-256, except for araJ. Bacteria developed resistance to antimicrobials by regulating various biological processes, among which the most involved is the cell wall synthesis (dacC, mrdA, lpxD, mrcB, and ftsI). The combination mutations of mrdA, envZ, and ftsI genes may increase the resistance to IMP. Our study could improve the understanding of the molecular mechanism of IMP resistance in E. coli.202235147175
233830.9693Characterization of disinfectant susceptibility profiles among clinical isolates of Acinetobacter baumannii in Ardabil, Iran. Antimicrobial disinfectants have been extensively used to control hospital-acquired infections worldwide. Prolonged exposure to bacteria could promote resistance to antimicrobial disinfectants. This study evaluated the antimicrobial activity of four commonly used disinfectants; triclosan, chlorhexidine digluconate, benzalkonium chloride, and formaldehyde against Acinetobacter baumannii clinical isolates. This study also determined the prevalence and association of efflux pumps encoding genes qacE, qacED1, emrA, and aceI with tolerance to disinfectants. A total of 100 A. baumannii isolates were included in the current study. The antimicrobial disinfectants' minimum inhibitory concentration (MIC) was determined using an agar dilution method. Genes involved in resistance to disinfectants were investigated by PCR method. The benzalkonium chloride MICs ranged between 32 and 128 μg mL-1, chlorhexidine digluconate 8-64 μg mL-1, triclosan 1-32 μg mL-1, and formaldehyde 128 μg mL-1. Overall, the highest MIC90 value was identified for formaldehyde (128 μg mL-1), followed by benzalkonium chloride and chlorhexidine digluconate (64 μg mL-1, each one) and triclosan (4 μg mL-1). In the present study, the qacE, qacED1, emrA, and aceI genes were found in 91%, 55%, 100%, and 88% of isolates, respectively. The qacG gene was not identified in our A. baumannii isolates. The qacED1 gene was associated with higher MICs for all disinfectants tested (P < 0.05), while the qacE and aceI genes were associated with higher MICs for benzalkonium chloride and chlorhexidine. This study indicated that triclosan is the most effective disinfectant against A. baumannii isolates.202338063878
245440.9692Colistin resistance in Gram-negative bacteria analysed by five phenotypic assays and inference of the underlying genomic mechanisms. BACKGROUND: Colistin is used against multi-drug resistant pathogens, yet resistance emerges through dissemination of plasmid-mediated genes (mcr) or chromosomal mutation of genes involved in lipopolysaccharide synthesis (i.e. mgrB, phoPQ, pmrCAB). Phenotypic susceptibility testing is challenging due to poor diffusion of colistin in agar media, leading to an underestimation of resistance. Performance of five phenotypic approaches was compared in the context of different molecular mechanisms of resistance. We evaluated Vitek 2® (bioMérieux, AST N242), Colistin MIC Test Strip (Liofilchem Diagnostici), UMIC (Biocentric), and Rapid Polymyxin™ NP test (ELITechGroup) against the standard broth microdilution (BMD) method. We used whole genome sequencing (WGS) to infer molecular resistance mechanisms. We analysed 97 Enterobacterales and non-fermenting bacterial isolates, largely clinical isolates collected up to 2018. Data was analysed by comparing susceptibility categories (susceptible or resistant) and minimal inhibitory concentrations (MIC). Susceptibility category concordance is the percentage of test results sharing the same category to BMD. MIC concordance was calculated similarly but considering ±1 MIC titre error range. We determined genomic diversity by core genome multi locus sequencing typing (cgMLST) and identified putative antimicrobial resistance genes using NCBI and CARD databases, and manual annotation. RESULTS: Of 97 isolates, 54 (56%) were resistant with standard BMD. Highest susceptibility category concordance was achieved by Rapid Polymyxin™ NP (98.8%) followed by UMIC (97.9%), Colistin E-test MIC strip (96.9%) and Vitek 2® (95.6%). Highest MIC concordance was achieved by UMIC (80.4%), followed by Vitek 2® (72.5%) and Colistin E-test MIC strip (62.9%). Among resistant isolates, 23/54 (43%) were intrinsically resistant to colistin, whereas 31/54 (57%) isolates had acquired colistin resistance. Of these, mcr-1 was detected in four isolates and mcr-2 in one isolate. Non-synonymous mutations in mgrB, phoQ, pmrA, pmrB, and pmrC genes were encountered in Klebsiella pneumoniae, Escherichia coli, and Acinetobacter bereziniae resistant isolates. Mutations found in mgrB and pmrB were only identified in isolates exhibiting MICs of ≥16 mg/L. CONCLUSIONS: The Rapid Polymyxin™ NP test showed highest categorical concordance and the UMIC test provided MIC values with high concordance to BMD. We found colistin resistance in diverse species occurred predominantly through spontaneous chromosomal mutation rather than plasmid-mediated resistance.202134798825
125150.9692Biofilm Formation and Plasmid-Mediated Quinolone Resistance Genes at Varying Quinolone Inhibitory Concentrations in Quinolone-Resistant Bacteria Superinfecting COVID-19 Inpatients. The likelihood of antimicrobial failure in COVID-19 patients with bacterial superinfection arises from both phenotypic (biofilms) and genotypic mechanisms. This cross-sectional study aimed to determine the inhibitory concentrations of quinolones-nalidixic acid, norfloxacin, ciprofloxacin, ofloxacin, and levofloxacin-in biofilm formers (minimum biofilm inhibitory concentration [MBIC]) and nonformers (minimum inhibitory concentration [MIC]) and correlate inhibitory concentrations with plasmid-mediated quinolone resistance (PMQR) genes in quinolone-resistant bacteria isolated from COVID-19 inpatients. Quinolone-resistant bacteria (n = 193), verified through disc diffusion, were tested for quinolone inhibitory concentrations using broth microdilution and biofilm formation using microtiter plate methods. The polymerase chain reaction was used to detect PMQR genes. Study variables were analyzed using SPSS v.17.0, with a significance level set at P <0.05. MIC-to-MBIC median fold increases for ciprofloxacin, ofloxacin, and levofloxacin were 128 (2-8,192), 64 (4-1,024), and 32 (4-512) in gram-positive cocci (GPC, n = 43), respectively, whereas they were 32 (4-8,192), 32 (4-2,048), and 16 (2-1,024) in fermentative gram-negative bacilli (F-GNB, n = 126) and 16 (4-4,096), 64 (2-64), and 16 (8-512) in nonfermentative gram-negative bacilli (NF-GNB, n = 24). In biofilm-forming F-GNB and NF-GNB, qnrB (10/32 versus 3/10), aac(6')-Ib-cr (10/32 versus 4/10), and qnrS (9/32 versus 0/10) genes were detected. A 32-fold median increase in the MIC-to-MBIC of ciprofloxacin was significantly (P <0.05) associated with qnrA in F-GNB and qnrS in NF-GNB. Biofilms formed by F-GNB and NF-GNB were significantly associated with the aac(6')-Ib-cr and qnrS genes, respectively. Nearly one-third of the superinfecting bacteria in COVID-19 patients formed biofilms and had at least one PMQR gene, thus increasing the need for quinolones at higher inhibitory concentrations.202539561392
229960.9692Determining the resistance of carbapenem-resistant Klebsiella pneumoniae to common disinfectants and elucidating the underlying resistance mechanisms. INTRODUCTION: Carbapenem-resistant Klebsiella pneumoniae (CRKP) infection is a serious problem in hospitals worldwide, posing a particular risk to immunocompromised patients. Elimination strategies may prevent these drug-resistant bacteria from spreading within hospital environments. Here, the susceptibility of patient-derived CRKP strains to common chemical disinfectants and possible correlations between the presence of drug-resistance genes and increased resistance to disinfectants were investigated. METHODS: The minimum inhibitory (MIC) and the minimum bactericidal concentrations (MBC) of common chemical disinfectants against each CRKP strain were determined using agar dilution; K. pneumoniae ATCC700603 served as a standard. The presence of the drug-resistance genes qacΔE, qacA, acrA and qacE was determined using PCR. RESULTS: A total of 27 clinically isolated CRKP strains collected in our hospital from 2011 to 2013 exhibited sensitivity to the following common chemical disinfectants in decreasing order of sensitivity: 75% ethyl alcohol > 2% glutaraldehyde > "84" disinfectant > 0.2% benzalkonium bromide > 2% iodine tincture > 1% iodophor > 0.1% chlorhexidine acetate. Of the 27 strains, 59, 41, 19 and 15% contained qacΔE, qacA, acrA and qacE resistance genes; 15% carried acrA, qacΔE and qacA, and 26% carried both qacA and qacΔE. Comparative analysis indicated that drug-resistance genes were correlated with higher MIC values. CONCLUSION: These pan-resistant pathogenic CRKP strains contained various drug-resistance genes and exhibited relatively high resistance to ethyl alcohol, chlorhexidine acetate and iodophor. Monitoring the drug-resistance rates of CRKP strains displaying disinfectant resistance may facilitate appropriate and effective sterilisation and thus preventing the spread of these pan-resistant strains.201526184804
233770.9690Klebsiella pneumoniae susceptibility to biocides and its association with cepA, qacΔE and qacE efflux pump genes and antibiotic resistance. BACKGROUND: Although antiseptics are some of the most widely used antibacterials in hospitals, there is very little information on reduced susceptibility to these biocides and its relationship with resistance to antibiotics. AIM: To determine the relationship between reduced susceptibility to biocides and the carriage of antiseptic resistance genes, cepA, qacΔE and qacE, as well as identifying the role of efflux pumps in conferring reduced susceptibility. METHODS: Susceptibility was assessed for five biocides: chlorhexidine, benzalkonium chloride, Trigene, MediHex-4, Mediscrub; and for 11 antibiotics against 64 isolates of Klebsiella pneumoniae. Susceptibility to all compounds was tested by the agar double dilution method (DDM) and the effect of efflux pumps on biocides determined by repeating the susceptibility studies in the presence of the efflux pump inhibitor carbonyl cyanide m-chlorophenyl hydrazone (CCCP). The presence of the cepA, qacΔE and qacE genes was identified by polymerase chain reaction. FINDINGS: The bacteria were not widely antibiotic resistant though a few showed reduced susceptibility to cefoxitin, chloramphenicol and rifampicin and later-generation cephalosporins but not to carbapenems. Biocide susceptibility, tested by DDM, showed that 50, 49 and 53 strains had reduced susceptibility to chlorhexidine, Trigene and benzalkonium chloride, respectively. The antiseptic resistance genes cepA, qacΔE and qacE were found in 56, 34 and one isolates respectively and their effects as efflux pumps were determined by CCCP (10 mg/L), which decreased the minimum inhibitory concentrations (MICs) of chlorhexidine and Medihex-4 by 2-128-fold but had no impact on the MICs of benzalkonium chloride, Trigene and Mediscrub. CONCLUSION: There was a close link between carriage of efflux pump genes, cepA, qacΔE and qacE genes and reduced biocide susceptibility, but not antibiotic resistance, in K. pneumoniae clinical isolates.201222498639
247980.9689Down-regulatory effects of green coffee extract on las I and las R virulence-associated genes in Pseudomonas aeruginosa. BACKGROUND: Antibiotic resistant strains of Pseudomonas aeruginosa are the cause of Gram negative nosocomial infections especially among the immunosuppressed patients. The bacteria contains las I and las R genes that play very important roles in the pathogenesis and mechanisms of aggression. These genes can be influenced by the quorum sensing (QS) system and such mechanism is becoming clinically important worldwide. This study aimed to investigate the preventive effects of green coffee extract (GCE) on the expression of pathogenesis-related genes, las I and las R in P. aeruginosa. METHODS: A total of fifty four P. aeruginosa strains were isolated out of 100 clinical samples collected from the infectious wards in different hospitals (Tehran province) using conventional microscopic and biochemical methods. Susceptibility of the isolates to different antibiotics, GCE and chlorogenic acid were elucidated. Multiplex polymerase chain reaction (PCR) and real-time PCR were performed to detect and quantify the expression levels of las I and las R genes. The presence of chlorogenic acid in GCE was confirmed by HPLC. RESULTS: Antibiotic susceptibility tests revealed multidrug resistance among the clinical isolates of those 40 strains were resistant to ciprofloxacin (74.07%), 43 to ceftazidime (79.26%), 29 to amikacin (53.7%), 42 to ampicillin (77.77%), 17 to colistin (31.48%), 40 to gentamicin (74.77%), and 50 to piperacillin (92.59%). PCR outcomes exhibited that the frequency of las I and las R genes were 100% in resistant and sensitive strains isolated from clinical and standard strains of P. aeruginosa (ATCC 15449). Real-time PCR analyses revealed that GCE significantly prevented the expression of las I and las R genes in P. aeruginosa. GCE at concentration level as low as 2.5 mg/mL could prevent the expression of lasI and lasR genes in P. aeruginosa clinical isolates. CONCLUSION: The presence and expression levels of las I and las R genes in P. aeruginosa isolates were investigated when the bacteria was exposed to GCE. Our results tend to suggest that genes involved in pathogenesis of:Pseudomonas aeruginosa are down regulated by quorum sensing effect of chlorogenic acid and therefore GCE could be useful as an adjuvant in combating multidrug resistance strains of Pseudomonas aeruginosa.201931187452
142890.9689Carbapenem-resistant Gram-negative bacteria associated with catheter-related bloodstream infections in three intensive care units in Egypt. We aimed to identify the carbapenem-resistant Gram-negative bacteria (GNB) causing catheter-related bloodstream infections (CRBSI) in intensive care units (ICU) in a tertiary care Egyptian hospital, to study their resistance mechanisms by phenotypic and genetic tests, and to use ERIC-PCR for assessing their relatedness. The study was conducted over 2 years in three ICUs in a tertiary care hospital in Egypt during 2015-2016. We identified 194 bloodstream infections (BSIs); 130 (67.01%) were caused by GNB, of which 57 were isolated from CRBSI patients (73.84%). Identification of isolates was performed using conventional methods and MALDI-TOF MS. Antimicrobial susceptibility testing (AST) was done by disc diffusion following CLSI guidelines. Phenotypic detection of carbapenemases enzymes activity was by modified Hodge test and the Carba-NP method. Isolates were investigated for the most common carbapenemases encoding genes bla(KPC), bla(NDM), and bla(OXA-48) using multiplex PCR. Molecular typing of carbapenem-resistant isolates was done by ERIC-PCR followed by sequencing of common resistance genes. The overall rate of CRBSI in our study was 3.6 per 1000 central venous catheter (CVC) days. Among 57 Gram-negative CRBSI isolates, Klebsiella pneumoniae (K. pneumoniae) was the most frequently isolated (27/57; 47.4%), of which more than 70% were resistant to Meropenem. Phenotypic tests for carbapenemases showed that 37.9% of isolates were positive by modified Hodge test and 63.8% by Carba-NP detection. Multiplex PCR assay detected the bla(NDM) in 28.6% of the isolates and bla(KPC) in 26.8%, bla(NDM) and bla(KPC) were detected together in the same isolate in 5.6%, while bla(OXA-48)-like were not detected. ERIC-PCR detected limited genetic relatedness between K. pneumoniae isolates. Elevated resistance rates were observed to all antibiotics including carbapenems among K. pneumoniae isolates causing CRBSI. ERIC-PCR showed that the resistant isolates were mainly polyclonal. Our results call for reinforcement of antimicrobial stewardship and measures to prevent CRBSI.201829936619
2298100.9689Burden of biocide resistance among multidrug-resistant bacteria isolated from various clinical specimens in a tertiary care hospital. BACKGROUND: Most studies on biocide resistance and its genetic determinants arise from environmental or food-borne microbial isolates and only a few from clinically relevant isolates. OBJECTIVES: This study determines the proportion of biocide resistance against five commonly used biocides and detects biocide resistance genes among MDR bacterial isolates using PCR. METHODS: Consecutive MDR isolates (n ​= ​180) were included (30 each of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Staphylococcus aureus, and Enterococcus species) from clinical specimens of various inpatient units at JIPMER. The isolates were challenged at 0.5,1 and 2 Macfarland (McF) inoculum with discrete dilutions of disinfectants. The minimum bactericidal concentrations (MBCs) for 70% Ethanol, 1.5% Cresol, 2% Glutaraldehyde, 1% Cetrimide, and 1% Chlorhexidine were determined for the isolates using ATCC reference strains as controls. PCR was performed targeting qac A/B, G; smr; and nfx B genes. RESULTS: For all biocides, MDR isolates had MBCs less than the maximum MBCs of ATCC strains. For MDR K. pneumoniae, A. baumannii, and P. aeruginosa, the highest MBCs of chlorhexidine and cetrimide were ≥75 and ​≥ ​150 ​μg/ml respectively at 0.5 McF inoculum; whereas these organisms grew at higher inoculum (2McF) even at commercially recommended biocidal concentration (1%) corresponding to 750 and 1500 ​μg/ml of chlorhexidine and cetrimide respectively. Meanwhile, the highest MBCs of MDR E. coli were 75 and 150 ​μg/ml for chlorhexidine and cetrimide respectively. Interestingly, the Gram-positive cocci survived the action of up to 35% ethanol. The nfxB and qacG genes were detected in 87% and 6.67% of MDR P. aeruginosa isolates respectively with no biocide resistance genes detected among the other organisms. CONCLUSIONS: Biocide dilutions challenged with higher inoculum indicated a narrow margin of effectiveness for certain biocides. Although a significant proportion of clinical MDR isolates of P. aeruginosa harbored biocide resistance genes, this finding had no phenotypic correlation.202337769586
1425110.9688Distribution and Antimicrobial Resistance of Complicated Intraabdominal Infection Pathogens in Two Tertiary Hospitals in Egypt. Background: Management of complicated intraabdominal infections (cIAIs) requires containment of the source and appropriate initial antimicrobial therapy. Identifying the local data is important to guide the empirical selection of antimicrobial therapy. In this study, we aimed to describe the pathogen distribution and antimicrobial resistance of cIAI. Methods: In two major tertiary care hospitals in Egypt, we enrolled patients who met the case definition of cIAI from October 2022 to September 2023. Blood cultures were performed using the BACTAlert system (BioMerieux, Marcy l'Etoile, France). A culture of aspirated fluid, resected material, or debridement of the infection site was performed. Identification of pathogens and antimicrobial susceptibility testing were conducted by the VITEK-2 system (BioMerieux, Marcy l'Etoile, France). Gram-negative resistance genes were identified by PCR and confirmed by whole bacterial genome sequencing using the Nextera XT DNA Library Preparation Kit and sequencing with the MiSeq Reagent Kit 600 v3 (Illumina, USA) on the Illumina MiSeq. Results: We enrolled 423 patients, 275 (65.01%) males. The median age was 61.35 (range 25-72 years). We studied 452 recovered bacterial isolates. Gram-negative bacteria were the vast majority, dominated by E. coli, followed by Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, and Proteus mirabilis (33.6%, 30.5%, 13.7%, 13%, and 5.4%, respectively). High rates of resistance were detected to third- and fourth-generation cephalosporins and fluoroquinolones. No resistance was detected to colistin. Resistance to amikacin and tigecycline was low among all isolates. Resistance to meropenem and ceftazidime/avibactam was moderate. ESBL genes were common in E. coli and K. pneumoniae. CTX-M15 gene was the most frequent. Among Enterobacterales, bla(OXA-48) and bla(NDM) were the most prevalent carbapenemase genes. Pseudomonas aeruginosa isolates harbored a wide variety of carbapenemase genes (OXA, NDM, VIM, SIM, GIM, SPM, IMP, AIM), dominated by metallo-beta-lactamases. In 20.6% of isolates, we identified two or more resistance genes. Conclusion: High resistance rates were detected to third- and fourth-generation cephalosporins and fluoroquinolones. Amikacin and tigecyclines were the most active antimicrobials. Our data call for urgent implementation of antimicrobial stewardship programs and reinforcement of infection control.202439172656
2288120.9688Resistance of Stenotrophomonas maltophilia to Fluoroquinolones: Prevalence in a University Hospital and Possible Mechanisms. OBJECTIVE: The purpose of this study was to investigate the clinical distribution and genotyping of Stenotrophomonas maltophilia, its resistance to antimicrobial agents, and the possible mechanisms of this drug resistance. METHODS: S. maltophilia isolates were collected from clinical specimens in a university hospital in Northwestern China during the period between 2010 and 2012, and were identified to the species level with a fully automated microbiological system. Antimicrobial susceptibility testing was performed for S. maltophilia with the Kirby-Bauer disc diffusion method. The minimal inhibitory concentrations (MICs) of norfloxacin, ofloxacin, chloramphenicol, minocycline, ceftazidime, levofloxacin and ciprofloxacin against S. maltophilia were assessed using the agar dilution method, and changes in the MIC of norfloxacin, ciprofloxacin and ofloxacin were observed after the addition of reserpine, an efflux pump inhibitor. Fluoroquinolone resistance genes were detected in S. maltophilia using a polymerase chain reaction (PCR) assay, and the expression of efflux pump smeD and smeF genes was determined using a quantitative fluorescent (QF)-PCR assay. Pulsed-field gel electrophoresis (PFGE) was employed to genotype identified S. maltophilia isolates. RESULTS: A total of 426 S. maltophilia strains were isolated from the university hospital from 2010 to 2012, consisting of 10.1% of total non-fermentative bacteria. The prevalence of norfloxacin, ciprofloxacin and ofloxacin resistance was 32.4%, 21.9% and 13.2% in the 114 S. maltophilia isolates collected from 2012, respectively. Following reserpine treatment, 19 S. maltophilia isolates positive for efflux pump were identified, and high expression of smeD and smeF genes was detected in two resistant isolates. gyrA, parC, smeD, smeE and smeF genes were detected in all 114 S. maltophilia isolates, while smqnr gene was found in 25.4% of total isolates. Glu-Lys mutation (GAA-AAA) was detected at the 151th amino acid of the gyrA gene, while Gly-Arg mutation (GGC-CGC) was found at the 37th amino acid of the parC gene. However, no significant difference was observed in the prevalence of gyrA or parC mutation between fluoroquinolone-resistant and -susceptible isolates (p> 0.05). The smqnr gene showed 92% to 99% heterogenicity among the 14 S. maltophilia clinical isolates. PFGE of 29 smqnr gene-positive S. maltophilia clinical isolates revealed 25 PFGE genotypes and 28 subgenotypes. CONCLUSIONS: Monitoring the clinical distribution and antimicrobial resistance of S. maltophilia is of great significance for the clinical therapy of bacterial infections. Reserpine is effective to inhibit the active efflux of norfloxacin, ciprofloxacin and ofloxacin on S. maltophilia and reduce MIC of fluoroquinolones against the bacteria. The expression of efflux pump smeD and smeF genes correlates with the resistance of S. maltophilia to fluoroquinolones.201525985315
1258130.9688Occurrence of antimicrobial resistance and antimicrobial resistance genes in methicillin-resistant Staphylococcus aureus isolated from healthy rabbits. BACKGROUND AND AIM: Methicillin-resistant globally, Staphylococcus aureus (MRSA) is a major cause of disease in both humans and animals. Several studies have documented the presence of MRSA in healthy and infected animals. However, there is less information on MRSA occurrence in exotic pets, especially healthy rabbits. This study aimed to look into the antimicrobial resistance profile, hidden antimicrobial-resistant genes in isolated bacteria, and to estimate prevalence of MRSA in healthy rabbits. MATERIALS AND METHODS: Two-hundreds and eighteen samples, including 42 eyes, 44 ears, 44 oral, 44 ventral thoracic, and 44 perineal swabs, were taken from 44 healthy rabbits that visited the Prasu-Arthorn Animal Hospital, in Nakornpathom, Thailand, from January 2015 to March 2016. The traditional methods of Gram stain, mannitol fermentation, hemolysis on blood agar, catalase test, and coagulase production were used to confirm the presence of Staphylococcus aureus in all specimens. All bacterial isolates were determined by antimicrobial susceptibility test by the disk diffusion method. The polymerase chain reaction was used to identify the antimicrobial-resistant genes (blaZ, mecA, aacA-aphD, msrA, tetK, gyrA, grlA, and dfrG) in isolates of MRSA with a cefoxitin-resistant phenotype. RESULTS: From 218 specimens, 185 S. aureus were isolated, with the majority of these being found in the oral cavity (29.73%) and ventral thoracic area (22.7%), respectively. Forty-seven (25.41%) MRSAs were found in S. aureus isolates, with the majority of these being found in the perineum (16, 34.04%) and ventral thoracic area (13, 27.66%) specimens. Among MRSAs, 29 (61.7%) isolates were multidrug-resistant (MDR) strains. Most of MRSA isolates were resistant to penicillin (100%), followed by ceftriaxone (44.68%) and azithromycin (44.68%). In addition, these bacteria contained the most drug-resistance genes, blaZ (47.83%), followed by gyrA (36.17%) and tetK (23.4%). CONCLUSION: This study revealed that MRSA could be found even in healthy rabbits. Some MRSAs strains were MDR-MRSA, which means that when an infection occurs, the available antibiotics were not effective in treating it. To prevent the spread of MDR-MRSA from pets to owners, it may be helpful to educate owners about effective prevention and hygiene measures.202236590129
1441140.9687Molecular characterisation of carbapenem-resistant Klebsiella pneumoniae clinical isolates: preliminary experience from a tertiary care teaching hospital in the Himalayas. BACKGROUND: There is a lack of whole-genome sequencing (WGS) data on multidrug-resistant (MDR) bacteria from the Uttarakhand region of India. The aim of this study was to generate WGS data of carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates recovered from patients in Uttarakhand's tertiary care centre. METHODS: A cross-sectional study included 29 MDR K. pneumoniae test isolates obtained from various clinical samples submitted to the bacteriology laboratory for culture and sensitivity testing from July 2018 to August 2019. After preliminary identification and antibiotic susceptibility testing, these isolates were subjected to WGS. RESULTS: A total of 27 of 29 isolates were CRKP. ST14 was the most common sequence type (n=8 [29.6%]). Carbapenem resistance was mainly encoded by OXA-48-like genes (21/27 [77.8%]). All isolates had a varied arsenal of resistance genes to different antibiotic classes. KL2 (9/27 [33.3%]) and KL51 (8/27 [29.6%]) were dominant K loci types. O1 and O2 together accounted for 88.9% (n=27) of CRKP isolates. Genes encoding yersiniabactin (ybt) and aerobactin (iuc) were identified in 88.9% (24/27) and 29.6% (8/27) of isolates. The predominant plasmid replicons present were ColKP3 (55.5%), IncFII(K) (51.8%) and IncFIB(pQil) (44.4%). CONCLUSIONS: This study emphasises the need for continued genomic surveillance of MDR bacteria that could be instrumental in developing treatment guidelines based on integrating phenotypic and molecular methods.202235029688
2189150.9687High prevalence of Panton-Valentine Leucocidin (PVL) toxin carrying MRSA and multidrug resistant gram negative bacteria in late onset neonatal sepsis indicate nosocomial spread in a Pakistani tertiary care hospital. BACKGROUND: Neonatal sepsis has high incidence with significant mortality and morbidity rates in Pakistan. We investigated common etiological patterns of neonatal sepsis at a tertiary care setup. METHODS: 90 pus and blood, gram negative and gram positive bacterial isolates were analyzed for virulence and antibiotic resistance gene profiling using PCR and disc diffusion methods. RESULTS: Staphylococcus aureus showed strong association with neonatal sepsis (43 %) followed by Citrobacter freundii (21 %), Pseudomonas aeruginosa (13 %), Escherichia coli (15 %) and Salmonella enterica (8 %). Molecular typing of E. coli isolates depicted high prevalence of the virulent F and B2 phylogroups, with 4 hypervirulent phylogroup G isolates. 76.9 % S. aureus isolates showed presence of Luk-PV, encoding for Panton-valentine leucocidin (PVL) toxin with majority also carrying MecA gene and classified as methicillin resistant S. aureus (MRSA). ecpA, papC, fimH and traT virulence genes were detected in E. coli and Salmonella isolates. 47 % Citrobacter freundii isolates carried the shiga like toxin SltII B. Antimicrobial resistance profiling depicted common resistance to cephalosporins, beta lactams and fluoroquinolones. CONCLUSION: Presence of PVL carrying MRSA and multidrug resistant gram negative bacteria, all isolated from late onset sepsis neonates indicate a predominant nosocomial transmission pattern which may complicate management of the disease in NICU setups.202336621204
2478160.9687Study on the resistance mechanism via outer membrane protein OprD2 and metal β-lactamase expression in the cell wall of Pseudomonas aeruginosa. The aim of the present study was to evaluate the imipenem-resistant mechanism via the outer membrane protein (OMP) OprD2 and metal β-lactamase expression in the cell wall of Pseudomonas aeruginosa. The Pseudomonas aeruginosa was clinically separated and validated by VITEK-2 full-automatic bacteria analyzer. Drug resistance, sensitive antibiotics and minimum inhibitory concentration (MIC) were tested using the drug sensitivity analysis system. The phenotype positive strains of MBL genes were screened using the Kirby-Bauer diffusion method by adding metal ion-chelating agent EDTA on the imipenem susceptibility paper. IMP-1, VIM-1 and SPM metaloenzyme genes were tested by polymerase chain reaction (PCR)-telomeric repeat amplification protocol (TRAP). The OMP OprD2 genes were tested by PCR-TRAP, and the protein expression was tested using western blot analysis. The location of OMP OprD2 was confirmed using the sodium salicylate inhibition test. The results showed that 80 portions (40%) of MBL-positive strains were screened out of 200 specimens. Imipenem-resistant Pseudomonas aeruginosa (IRPA) and MIC values were significantly higher than quality control bacteria and control bacteria (P<0.05). A total of 35 cases with IMP-1 positive, 20 with VIM-1 positive, 16 with SPM positive, 5 with 2 positive genes and 4 with 3 positive genes were screened among MBL positive strains. A total of 150 portions (75%) of OprD2 deficiencies were screened from 200 specimens. The standard strains and sensitive strains showed OprD2 protein bands at 45 kDa while no OprD2 protein bands appeared in OprD2 deficiency strains. It was in accordance with gene detection. In conclusion, OMP OprD2 deficiency and MBL phenotype positivity may be important mechanisms of IRPA.201627882088
1212170.9686Virulence Factors and Antimicrobial Resistance of Uropathogenic Escherichia coli EQ101 UPEC Isolated from UTI Patient in Quetta, Balochistan, Pakistan. Infectious diseases have been tremendously increasing as the organisms of even normal flora become opportunistic and cause an infection, and Escherichia coli (E. coli EQ101) is one of them. Urinary tract infections are caused by various microorganisms, but Escherichia coli is the primary cause of almost 70%-90% of all UTIs. It has multiple strains, possessing diverse virulence factors, contributing to its pathogenicity. Furthermore, these virulent strains also can cause overlapping pathogenesis by sharing resistance and virulence factors among each other. The current study is aimed at analyzing the genetic variants associated with multi-drug-resistant (MDR) E. coli using the whole genome sequencing platform. The study includes 100 uropathogenic Escherichia coli (UPEC) microorganisms obtained from urine samples out of which 44% were multi-drug-resistant (MDR) E. coli. Bacteria have been isolated and antimicrobial susceptibility test (AST) was determined by disk diffusion method on the Mueller-Hinton agar plate as recommended by the Clinical and Laboratory Standards Institute (CLSI) 2020, and one isolate has been selected which shows resistance to most of the antibiotics, and that isolate has been analyzed by whole genome sequencing (WGS), accompanied by data and phylogenetic analysis, respectively. Organisms were showing resistance against ampicillin (10 μg), cefixime (5 μg), ceftriaxone (30 μg), nalidixic acid (30 μg), ciprofloxacin (5 μg), and ofloxacin (5 μg) on antimicrobial susceptibility test. WGS were done on selected isolate which identified 25 virulence genes (air, astA, chuA, fyuA, gad, hra, iha, irp2, iss, iucC, iutA, kpsE, kpsMII_K1, lpfA, mchF, ompT, papA_F43, sat, senB, sitA, terC, traT, usp, vat, and yfcV) and seven housekeeping genes (adk, fumC, gyrB, icd, mdh, purA, and recA). Among resistance genes, seven genes (TolC, emrR, evgA, qacEdelta1, H-NS, cpxA, and mdtM) were identified to be involved in antibiotic efflux, three AMR genes (aadA5, mphA, and CTX-M-15) were involved in antibiotic inactivation, and two genes (sul1 and dfrA14) were found to be involved in antibiotic drug replacement. Our data identified antibiotic resistance and virulence genes of the isolate. We suggest further research work to establish region-based resistance profile in comparison with the global resistance pattern.202337727279
1331180.9685Serotypes, antibiotic resistance, and virulence genes of Salmonella in children with diarrhea. BACKGROUND: Salmonella is an important foodborne pathogen that causes acute diarrhea in humans worldwide. This study analyzed the relationships of serotypes and antibiotic resistance with virulence genes of Salmonella isolated from children with salmonellosis. METHODS: Serological typing was performed using the slide-agglutination method. The Kirby-Bauer disk diffusion method was used to test antibiotic susceptibility. Twenty virulence genes were detected by PCR. RESULTS: Salmonella Typhimurium (21 isolates, 34.43%) and S Enteritidis (12 isolates, 19.67%) were the predominant species among the 61 isolates. Ampicillin resistance was most common (63.93%), and among the cephalosporins, resistance was most often found to cefotaxime, a third-generation cephalosporin (19.67%). Among the 20 virulence genes, prgH, ssrB, and pagC were detected in all Salmonella isolates. In S Typhimurium, the detection rates of hilA, sipB, marT, mgtC, sopB, pagN, nlpI, bapA, oafA, and tolC were high. In S Enteritidis, the detection rates of icmF, spvB, spvR, and pefA were high. Nitrofurantoin resistance was negatively correlated with the virulence gene bapA (P = .005) and was positively correlated with icmF, spvB, spvR, and pefA (P = .012, .008, .002, and .005, respectively), The P values between all other virulence genes and antibiotic resistance were >.05. CONCLUSION: Salmonella Typhimurium and S Enteritidis were the main serotypes in children with diarrhea in Hangzhou, China. Salmonella exhibited a high level of resistance to common antibiotics, and a high rate of bacteria carrying virulence genes was observed. However, no significant correlation was found between virulence genes and resistance to common antibiotics.202032797660
2300190.9685Determining the Disinfectants Resistance Genes and the Susceptibility to Common Disinfectants of Extensively Drug-Resistant Carbapenem-Resistant Klebsiella pneumoniae Strains at a Tertiary Hospital in China. Carbapenem-resistant Klebsiella pneumoniae (CRKP) infection has become a significant threat to global health. The application of chemical disinfectants is an effective infection control strategy to prevent the spread of CRKP in hospital environments. However, bacteria have shown reduced sensitivity to clinical disinfectants in recent years. Furthermore, bacteria can acquire antibiotic resistance due to the induction of disinfectants, posing a considerable challenge to hospital infection prevention and control. This study collected 68 CRKP strains from the Fifth Affiliated Hospital of Xinjiang Medical University in China from 2023 to 2024. These strains were isolated from the sputum, urine, and whole blood samples of patients diagnosed with CRKP infection. Antibiotic susceptibility tests were performed on CRKP strains. Concurrently, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of disinfectants (benzalkonium bromide, 1% iodophor disinfectant, alcohol, and chlorine-containing disinfectant) against the test isolates were determined by the broth microdilution method. The efflux pump genes (cepA, qacE, qacEΔ1, qacEΔ1-SUL1, oqxA, and oqxB) were detected using polymerase chain reaction. The results showed that 21 out of the 68 CRKP strains exhibited extensive drug resistance, whereas 47 were nonextensively drug-resistant. The MIC value for benzalkonium bromide disinfectants displayed statistically significant differences (p < 0.05) between extensively drug-resistant (XDR) and non-XDR strains. Additionally, the MBC values for benzalkonium bromide disinfectants and 1% iodophor disinfectants displayed statistically significant differences (p < 0.05) between XDR and non-XDR strains. The detection rates for the efflux pump genes were as follows: cepA 52.9%, qacE 39.7%, qacEΔ1 35.2%, qacEΔ1-SUL1 52.9%, oqxA 30.8%, and oqxB 32.3%. The detection rate of the qacEΔ1-SUL1 gene in XDR CRKP strains was significantly higher than in non-XDR CRKP strains (p < 0.05). This indicates a potential link between CRKP bacterial disinfectant efflux pump genes and CRKP bacterial resistance patterns. Ongoing monitoring of the declining sensitivity of XDR strains against disinfectants is essential for the effective control and prevention of superbug.202439166283