Increase In Minimum Inhibitory Concentration Research Articles

A greater effect on clarithromycin resistance of mef(A)-associated msr(D) than mef(E)-associated msr(D) in Streptococcus pyogenes.

The mef(A)- and its subclass mef(E) systems had long been considered to constitute one of the primary macrolide-resistant mechanisms in Streptococcus pyogenes. However, we have previously demonstrated that the msr(D) gene located immediately downstream of the mef(A)/mef(E) genes plays a predominant role in these systems. In previous studies, furthermore, mef(A)-associated msr(D)10-85 of an S. pyogenes strain (10-85) exhibited a greater increase in clarithromycin minimum inhibitory concentration (MIC) than mef(E)-associated msr(D)13-O-10 of another strain (13-O-10). Both msr(D) genes encode 487 amino acid residues, 13 amino acid residues of which are different from each other. In this study, we performed mutational analysis of the msr(D) genes and showed that a single-nucleotide polymorphism to cause a substitution of Asp238 with Gly is mainly associated with the greater increase in clarithromycin MIC by the msr(D)10-85 than by the msr(D)13-O-10 allele. In addition, another substitution of Ser with Arg at codon 194 is partially associated with the greater increase by the msr(D)10-85 than by the msr(D)13-O-10 allele.

In Vitro Evaluation of Resistance Development to Silver Sulfadiazine and Subsequent Cross-Resistance to Antibiotics.

Treatment of chronic wounds that are at risk of infection, or that are infected, require the use of antimicrobial dressings, most often those that contain silver. Silver exerts its antimicrobial effects by binding to multiple cellular components and, as such, bacterial resistance to it is low; however, molecular silver resistance has been documented and is attributed to the presence of the sil operon or changes in genes encoding porin and efflux pump expression. The aim of this study was to evaluate spontaneous silver resistance development in common opportunistic pathogens, Staphylococcus, Pseudomonas and Enterococcus cloacae, as well as resistance development when exposed to subtherapeutic concentrations over a prolonged period. Furthermore, following silver resistance development, cross-resistance to several classes of antibiotics was evaluated. Following exposure of the strains to silver sulfadiazine (SSD) at two times and four times minimum inhibitory concentration (MIC), the mutation rate was <1010 colony forming unit (CFU)/mL. Serial passage of S. aureus and P. aeruginosa in subinhibitory concentrations of SSD selected for no resistant mutants. The SSD MIC of E. cloacae increased past the solubility limit of SSD at serial passage 17. MIC testing of this isolate showed a >2048-fold increase in MIC to silver in comparison to the parent strain. MIC testing of the serial passage isolates demonstrated no cross-resistance to antibiotics from six different classes. Overall, the results of this study show resistance development to silver is low and, if it does occur, it does not confer resistance to several antibiotic classes. However, as this study was carried out with a small number of strains, a study with a larger panel of strains and sequencing of the strains to determine the exact mechanism of resistance would be needed to investigate the threat of silver resistance further.

The dlt operon contributes to the resistance to chlorhexidine in Streptococcus mutans

Chlorhexidine (CHX) is considered to be the gold standard for dental caries prevention and is widely applied in dental practice. However, the long-term application of CHX may result in CHX-resistance in oral pathogens. The aim of this study was to investigate whether long-term use of CHX causes resistance in Streptococcus mutans and to explore the possible associated mechanisms. Four different S. mutans strains were chosen for this study to exclude the specificity of strains. The four strains displayed an increase in minimum inhibitory concentration (MIC) after exposure to CHX for 10 passages. The features and cariogenicity of S. mutans CHX-resistant strains (SM-Cs) that were exposed to CHX for 10 passages with increased MIC did not differ significantly to the parental strains. The SM-Cs were more hydrophobic than the parental strains. The dltC and dltD genes were upregulated in SM-Cs. Relative expression of the BceA, BceR, and SMU.862 genes in SM-Cs was similar to or lower than that of the parental strains. The MIC value was significantly lower in dltC knockout mutants. These findings confirmed that continuous exposure to CHX could induce CHX-resistance in S. mutans. The increased cell surface hydrophobicity and upregulated expression of dlt operon were possible underlying mechanisms of CHX-resistance in S. mutans.

Novel Arginine End-Tagging Antimicrobial Peptides to Combat Multidrug-Resistant Bacteria.

The emergence of multidrug-resistant microorganisms has been termed one of the most common global health threats, emphasizing the discovery of new antibacterial agents. To address this issue, we engineered peptides harboring "RWWWR" as a central motif plus arginine (R) end-tagging and then tested them in vitro and in vivo. Our results demonstrate that Pep 6, one of the engineered peptides, shows great potential in combating Escherichia coli bacteremia and the Staphylococcus aureus skin burn infection model, which induces a 62-90% reduction in bacterial burden. Remarkably, after long serial passages of S. aureus and E. coli for 30 days, Pep 6 is still highly efficient in killing pathogens, compared with 64- and 128-fold increase in minimal inhibitory concentrations (MICs) for vancomycin and polymyxin B, respectively. We also found that Pep 6 exhibited robust biofilm-inhibiting activity and eliminated 61.33% of the mature methicillin-resistant Staphylococcus aureus (MRSA) biofilm with concentration in the MIC level. These results suggest that the RWWWR motif and binding of arginine end-tagging could be harnessed as a new agent for combating multidrug-resistant bacteria.

Sub-Inhibitory Concentrations of Chlorhexidine Induce Resistance to Chlorhexidine and Decrease Antibiotic Susceptibility in Neisseria gonorrhoeae.

Objectives: Chlorhexidine digluconate (chlorhexidine) and Listerine® mouthwashes are being promoted as alternative treatment options to prevent the emergence of antimicrobial resistance in Neisseria gonorrhoeae. We performed in vitro challenge experiments to assess induction and evolution of resistance to these two mouthwashes and potential cross-resistance to other antimicrobials.Methods: A customized morbidostat was used to subject N. gonorrhoeae reference strain WHO-F to dynamically sustained Listerine® or chlorhexidine pressure for 18 days and 40 days, respectively. Cultures were sampled twice a week and minimal inhibitory concentrations (MICs) of Listerine®, chlorhexidine, ceftriaxone, ciprofloxacin, cefixime and azithromycin were determined using the agar dilution method. Isolates with an increased MIC for Listerine® or chlorhexidine were subjected to whole genome sequencing to track the evolution of resistance.Results: We were unable to increase MICs for Listerine®. Three out of five cultures developed a 10-fold increase in chlorhexidine MIC within 40 days compared to baseline (from 2 to 20 mg/L). Increases in chlorhexidine MIC were positively associated with increases in the MICs of azithromycin and ciprofloxacin. Low-to-higher-level chlorhexidine resistance (2–20 mg/L) was associated with mutations in NorM. Higher-level resistance (20 mg/L) was temporally associated with mutations upstream of the MtrCDE efflux pump repressor (mtrR) and the mlaA gene, part of the maintenance of lipid asymmetry (Mla) system.Conclusion: Exposure to sub-lethal chlorhexidine concentrations may not only enhance resistance to chlorhexidine itself but also cross-resistance to other antibiotics in N. gonorrhoeae. This raises concern regarding the widespread use of chlorhexidine as an oral antiseptic, for example in the field of dentistry.

The Effect of Triclosan Adaptation on Antimicrobial Resistance among Clinical Escherichia coli Isolates from Egyptian Patients

There is a possible link between exposure to Triclosan (TCS) and changes in antimicrobial susceptibility. The change in the tolerance of clinical Escherichia coli (n=45) isolates to the biocide TCS, changes in antibiotic resistance and differences in the efflux pump mechanism were analyzed. 45 E. coli isolates were obtained. The minimum inhibitory concentration (MIC), the minimum bactericidal concentration (MBC) of TCS, and the expression of four efflux pump encoding genes in antibiotic-resistant isolates were determined before and after TCS adaptation. The number of TCS-tolerant isolates was 11 (24.4%). After adaptation, the percentage of tolerant isolates increased to 42.2% (n=19). A significant change (p&lt;0.05) in antimicrobial resistance of the tested isolates (n=45) before and after TCS adaptation was detected for ceftazidime, ceftriaxone, ertapenem, imipenem, amikacin, gentamicin, tobramycin, ciprofloxacin, levofloxacin and doxycycline. Among the new TCS tolerant isolates (n=8). there was an increase in TCS MIC as well as the MBC after TSC adaptation. The adapted isolates exhibited a significant increase in the expression of mdfA and norE genes (p=&lt;0.001). There is a strong correlation between efflux pump gene overexpression and susceptibility to TCS and other antimicrobials.

Emergence of OXA-833 in Proteus Species at a Tertiary Care Hospital in Dhaka, Bangladesh.

Context:Proteus species are liable for multitude of infections and associated with resistance to routinely used antibiotics even to reserve drugs such as carbapenems.Aims:The aim of this study was to detect the presence of MBL producers, including blaOXA-833 gene in Proteus spp. along with their antibiotic resistance pattern.Settings and Design:This cross-sectional study was conducted in the Department of Microbiology of a tertiary care hospital of Bangladesh during July 2018 to June 2019.Subjects and Methods:Proteus spp. was isolated from a total of 500 samples. Antibiotic susceptibility was performed by disk-diffusion technique. Minimum inhibitory concentration (MIC) of imipenem was determined by agar dilution method. Carbapenemase producers were phenotypically detected by double disc synergy (DDS) test, combined disc (CD) assay, and modified Hodge test (MHT). Carbapenemase genes (blaKPC, blaVIM, blaIMP, blaNDM-1, blaOXA-23, blaOXA-48-like/blaOXA-833, and blaOXA-58) among imipenem-resistant Proteus spp. were detected by polymerase chain reaction (PCR). Sequencing was performed to differentiate OXA-833 from OXA-48-like gene by capillary method, and the nucleotide sequence of OXA-833 has been deposited to GenBank.Results:Ten (25%) imipenem-resistant isolates were detected during disk-diffusion technique, among them 60%, 70%, 50% carbapenemase producers were detected by DDS test, CD assay, MHT, respectively, and 70% by PCR. A significant increase in MIC was found between 8 and ≥128 μg/ml to imipenem. PCR revealed that 40% imipenem-resistant isolates were positive for blaNDM-1 and blaVIM followed by 20% for blaOXA-48-like/blaOXA-833 and blaOXA-23, respectively. Sequencing of blaOXA-48-like gene established the OXA-833 variant of class D carbapenemase encoding gene.Conclusion:The results of this study showed the presence of high proportion of carbapenemase enzyme-producing Proteus spp. in Bangladesh. blaOXA-833 is emerging in Bangladesh.

Minimum inhibitory concentrations of commercial essential oils against common chicken pathogenic bacteria and their relationship with antibiotic resistance.

AimsWe investigated the antibacterial effect of seven essential oils (EOs) and one EO‐containing liquid phytogenic solution marketed for poultry and pigs (‘Product A’) on chicken pathogens, as well as the relationship between minimum inhibitory concentration (MIC) in EOs and antibiotics commonly administered to chicken flocks in the Mekong Delta (Vietnam).Methods and ResultsMicellar extracts from oregano (Origanum vulgare), cajeput (Melaleuca leucadendra), garlic (Allium sativum), black pepper (Piper nigrum), peppermint (Mentha × piperita L.), tea tree (Melaleuca alternifolia), cinnamon (Cinnamomum zeylanicum) EOs and Product A were investigated for their MIC against Avibacterium endocarditidis (N = 10), Pasteurella multocida (N = 7), Ornitobacterium rhinotracheale (ORT) (N = 10), Escherichia coli (N = 10) and Gallibacterium anatis (N = 10). Cinnamon EO had the lowest median MIC across strains (median 0.5 mg/ml [IQR, interquartile range 0.3–2.0 mg/ml]), followed by Product A (3.8 mg/ml [1.9–3.8 mg/ml]), oregano EO (30.4 mg/ml [7.6–60.8 mg/ml]) and garlic 63.1 mg/ml [3.9 to >505.0 mg/ml]. Peppermint, tea tree, cajeput and pepper EOs had all MIC ≥219 mg/ml. In addition, we determined the MIC of the 12 most commonly used antibiotics in chicken flocks in the area. After accounting for pathogen species, we found an independent, statistically significant (p < 0.05) positive correlation between MIC of 10 of 28 (35.7%) pairs of EOs. For 67/96 (69.8%) combinations of EOs and antibiotics, the MICs were correlated. Of all antibiotics, doxycycline was positively associated with the highest number of EOs (peppermint, tea tree, black pepper and cajeput, all p < 0.05). For cinnamon, the MICs were negatively correlated with the MICs of 11/12 antimicrobial tested (all except colistin).ConclusionsIncreases in MIC of antibiotics generally correlates with increased tolerance to EOs. For cinnamon EO, however, the opposite was observed.Significance and Impact of the StudyOur results suggest increased antibacterial effects of EOs on multi‐drug resistant pathogens; cinnamon EO was particularly effective against bacterial poultry pathogens.

Hybrid derivative of cathelicidin and human beta defensin-2 against Gram-positive bacteria: A novel approach for the treatment of bacterial keratitis

Bacterial keratitis (BK) is a major cause of corneal blindness globally. This study aimed to develop a novel class of antimicrobial therapy, based on human-derived hybrid host defense peptides (HyHDPs), for treating BK. HyHDPs were rationally designed through combination of functional amino acids in parent HDPs, including LL-37 and human beta-defensin (HBD)-1 to -3. Minimal inhibitory concentrations (MICs) and time-kill kinetics assay were performed to determine the concentration- and time-dependent antimicrobial activity and cytotoxicity was evaluated against human corneal epithelial cells and erythrocytes. In vivo safety and efficacy of the most promising peptide was examined in the corneal wound healing and Staphylococcus aureus (ATCC SA29213) keratitis murine models, respectively. A second-generation HyHDP (CaD23), based on rational hybridization of the middle residues of LL-37 and C-terminal of HBD-2, was developed and was shown to demonstrate good efficacy against methicillin-sensitive and methicillin-resistant S. aureus [MIC = 12.5–25.0 μg/ml (5.2–10.4 μM)] and S. epidermidis [MIC = 12.5 μg/ml (5.2 μM)], and moderate efficacy against P. aeruginosa [MIC = 25-50 μg/ml (10.4–20.8 μM)]. CaD23 (at 25 μg/ml or 2× MIC) killed all the bacteria within 30 min, which was 8 times faster than amikacin (25 μg/ml or 20× MIC). After 10 consecutive passages, S. aureus (ATCC SA29213) did not develop any antimicrobial resistance (AMR) against CaD23 whereas it developed significant AMR (i.e. a 32-fold increase in MIC) against amikacin, a commonly used treatment for BK. Pre-clinical murine studies showed that CaD23 (0.5 mg/ml) achieved a median reduction of S. aureus bioburden by 94% (or 1.2 log10 CFU/ml) while not impeding corneal epithelial wound healing. In conclusion, rational hybridization of human-derived HDPs has led to generation of a potentially efficacious and safe topical antimicrobial agent for treating Gram-positive BK, with no/minimal risk of developing AMR.

Silver nanoparticles–chitosan composites activity against resistant bacteria: tolerance and biofilm inhibition

This study aimed to evaluate the effectiveness of silver nanoparticles–chitosan composites (AgNPs) with different morphologies and particle size distributions against resistant bacteria and biofilm formation. Four different samples were prepared by a two-step procedure using sodium borohydride and ascorbic acid as reducing agents and characterized by UV–Vis absorption spectra, scanning transmission electron microscopy. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the AgNPs were determined according to the Clinical and Laboratory Standards Institute (CLSI) against clinical isolates multidrug-resistant and strains of the American Type Culture Collection (ATCC). An assay was performed to determine the MICs during 20 successive bacteria exposures to AgNPs to investigate whether AgNPs induce tolerance in bacteria. The antibiofilm activities of AgNPs were also evaluated by determining the minimum biofilm inhibitory concentration (MBIC). The spherical AgNPs present diameters ranging from 9.3 to 62.4 nm, and some samples also have rod-, oval-, and triangle-shaped nanoparticles. The MIC and MBC values ranged from 0.8 to 25 μg/mL and 3.1 to 50 μg/mL, respectively. Smaller and spherical AgNPs exhibited the highest activity, but all the AgNPs developed in this study exhibit bactericidal activity. There was no significant MIC increase after 20 passages to the AgNPs. Regarding the antibiofilm activity, MBICs ranged from 12.5 to 50 μg/mL. Again, smaller and spherical nanoparticles presented the best results with phenotypic inhibition of production of slime or exopolysaccharide (EPS) matrix. Thus, it was concluded that AgNPs have a promising potential against resistant bacteria and bacteria that grow on biofilms without inducing tolerance.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11051-021-05314-1.

The Prevalence and Antimicrobial Susceptibility Pattern of Gram-Positive Pathogens: Three-Year Study at a Tertiary Care Hospital in Mumbai, India

Introduction The growing resistance pattern of the gram-positive pathogens along with a steady increase in minimum inhibitory concentration of the currently available antibiotics have led to an increase in morbidity and mortality rates in India. This study aims to access the shifting antibiotic susceptibility paradigm of the gram-positive pathogens in various infections at a tertiary care center. Methods This is a 3-year retrospective observational study which was performed from January 2016 to December 2018 at a tertiary care hospital in Mumbai. All clinically significant gram-positive cocci isolated from a variety of clinical specimens were studied for their prevalence and antimicrobial susceptibility. Results Out of 4,428 gram-positive isolates, Staphylococcus aureus (35.3%) was the commonly encountered pathogen, followed by Enterococcus spp. (32.1%) and coagulase-negative Staphylococcus (CoNS) (25.7%). S. aureus was majorly isolated from skin and soft tissue infections (60.3%), followed by patients with respiratory tract infections (18.2%) and blood stream infections (13%). Among S. aureus , particularly methicillin-resistant S. aureus (MRSA), prevalence increased from 29.5% in 2016 to 35.1% in 2018, with an overall prevalence of 33.6%. All S. aureus isolates were 100% sensitive toward vancomycin, linezolid, tigecycline, and teicoplanin. However, the CoNS isolates showed a higher resistance rate with reduced susceptibility toward linezolid and teicoplanin. High prevalence of resistance was observed across gram-positive isolates with commonly used antibiotics such as ciprofloxacin, levofloxacin, and erythromycin. While the prevalence of linezolid-resistant enterococcus (LRE) was 3.6%, vancomycin (VRE) and teicoplanin resistance among the enterococcus species was as high as 7.7% and 7.5%, respectively. Conclusion Rising methicillin resistance among the Staphylococcal species (MRSA and MR-CoNS) along with reduced susceptibility toward currently available anti-MRSA agents is a matter of serious concern as it limits the therapeutic options for treating multidrug resistant (MDR) gram-positive infections.

Chlorhexidine leads to the evolution of antibiotic-resistant Pseudomonas aeruginosa.

Antimicrobial resistance is a major public-health concern. We evaluate chlorhexidine role in selection of resistant Pseudomonas aeruginosa mutants and their antibiotic cross-resistance. Mutation frequency and mutation rate after short-term exposure to sub-inhibitory concentrations of chlorhexidine were compared to those after spontaneous chlorhexidine-exposure, in P. aeruginosa PAO1 strain. Chlorhexidine-resistant mutants were generated, either by serial passage in increasing chlorhexidine concentrations or by single exposure to lethal chlorhexidine concentration. The generated mutants were tested for cross-resistance to different antibiotics, by determination of minimum inhibitory concentrations (MIC). The accompanied phenotypic changes in membrane permeability, outer membrane proteins (OMP), and efflux function were evaluated. The effect of exposure to chlorhexidine on MexAB-OprM, MexEF-oprN, and MexXY efflux pumps expression was investigated. No significant change was recorded between the mutation frequencies and mutation rates after short-term exposure to sub-inhibitory concentrations of chlorhexidine and after spontaneous chlorhexidine-exposure, in P. aeruginosa PAO1 strain. Twelve stable mutants, with ≥ eight-fold increase in chlorhexidine MIC, were generated. Several mutants showed increase in the MIC of colistin, cefepime, ceftazidime, meropenem, ciprofloxacin, and amikacin; seven mutants expressed meropenem cross-resistance. This was accompanied by decreased outer membrane permeability and changes in OMP. Using efflux pump inhibitor, chlorhexidine resistance was reverted in most isolates. Exposure to sub-inhibitory concentration of chlorhexidine induced the expression of MexXY efflux pump. Some resistant mutants had overexpressed MexXY efflux pump. Chlorhexidine can select P. aeruginosa strains with antibiotic cross-resistance. This necessitates implementing special protocols for chlorhexidine use and re-evaluation of its benefit versus risk in personal-care products.

Cefiderocol Activity Against Clinical Pseudomonas aeruginosa Isolates Exhibiting Ceftolozane-Tazobactam Resistance.

BackgroundMutations in the AmpC-AmpR region are associated with treatment-emergent ceftolozane-tazobactam (TOL-TAZ) and ceftazidime-avibactam (CAZ-AVI) resistance. We sought to determine if these mutations impact susceptibility to the novel cephalosporin-siderophore compound cefiderocol.MethodsThirty-two paired isolates from 16 patients with index P. aeruginosa isolates susceptible to TOL-TAZ and subsequent P. aeruginosa isolates available after TOL-TAZ exposure from January 2019 to December 2020 were included. TOL-TAZ, CAZ-AVI, imipenem-relebactam (IMI-REL), and cefiderocol minimum inhibitory concentrations (MICs) were determined using broth microdilution. Whole-genome sequencing of paired isolates was used to identify mechanisms of resistance to cefiderocol that emerged, focusing on putative mechanisms of resistance to cefiderocol or earlier siderophore-antibiotic conjugates based on the previously published literature.ResultsAnalyzing the 16 pairs of P. aeruginosa isolates, ≥4-fold increases in cefiderocol MICs occurred in 4 of 16 isolates. Cefiderocol nonsusceptibility criteria were met for only 1 of the 4 isolates, using Clinical and Laboratory Standards Institute criteria. Specific mechanisms identified included the following: AmpC E247K (2 isolates), MexR A66V and L57D (1 isolate each), and AmpD G116D (1 isolate) substitutions. For both isolates with AmpC E247K mutations, ≥4-fold MIC increases occurred for both TOL-TAZ and CAZ-AVI, while a ≥4-fold reduction in IMI-REL MICs was observed.ConclusionsOur findings suggest that alterations in the target binding sites of P. aeruginosa–derived AmpC β-lactamases have the potential to reduce the activity of 3 of 4 novel β-lactams (ie, ceftolozane-tazobactam, ceftazidime-avibactam, and cefiderocol) and potentially increase susceptibility to imipenem-relebactam. These findings are in need of validation in a larger cohort.

Evaluation of Changes Induced in the Probiotic Escherichia coli M17 Following Recurrent Exposure to Antimicrobials

Introduction: It is already well known that the exposure of certain bacteria, pathogenic or not, to antimicrobials is likely to increase their virulence and induce the development of direct or cross resistance to antimicrobials, but there is almost no information available regarding probiotics.&#x0D; Aim: To assess the changes induced in susceptibility to antibiotics, biofilm formation, growth rate and relative pathogenicity in the probiotic Escherichia coli M17 (EC-M17) after long exposure to antimicrobials namely ampicillin, kanamycin, cefazolin and silver nanoparticles (AgNPs).&#x0D; Methods: After determining the minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of the 4 antimicrobials above-mentioned by the microdilution method, EC-M17 was exposed to increasing subinhibitory doses ranging from MIC/8 to MIC for 8 days. The susceptibility to antibiotics of the mutants obtained was assessed by the Kirby Bauer disc diffusion method, biofilm formation by the Congo red agar method and with crystal violet bacterial attachment assay, and relative pathogenicity was assessed using a Galleria melonella waxworm model.&#x0D; Results: Exposure to antimicrobials induces noticeable changes in EC-M17. The highest adaptation to antimicrobials was observed on AgNPs with 8-fold increase in MIC and 16-fold increase in MBC of AgNPs. EC-M17 exposed to ampicillin, kanamycin and silver nanoparticles became resistant to ampicillin, ceftazidime, ceftazidime/clavulanate and tetracycline while exposure to cefazolin induced a significant decrease in sensitivity to tetracycline and ampicillin and resistance to ceftazidime/clavulanate and ceftazidime. The strain exposed to ampicillin was the only one to produce more biofilm than the control strain and except the EC-M17 exposed to cefazolin, all other EC-M17 strains were more pathogenic on G. melonella model than the control.&#x0D; Conclusion: Data in this investigation suggest that repeated exposure of the probiotic EC-M17 to antimicrobials may induce changes in antimicrobials susceptibility, biofilm formation, growth rate, and relative pathogenicity. Therefore, as far as possible, the probiotic E. coli M17 should not be used in combination with antibiotics and further investigations are required to expand similar work on more probiotics in order to avoid resistance build-up which might be transmitted by horizontal transfer.

Resistance elicited by sub-lethal concentrations of ampicillin is partially mediated by quorum sensing in Pseudomonas aeruginosa.

The rapid increase of antibiotic resistance is a serious challenge around the world. Antibiotics are present in various environments at sub-lethal concentrations, but how resistance emerges under sub-lethal conditions is not fully clear. In this study, we evolved Pseudomonas aeruginosa PAO1 under sub-lethal conditions, in the presence of either 15-30μg/mL or 150-300μg/mL of ampicillin. We found a~5-6 fold increase in the minimum inhibitory concentration (MIC) among evolved isolates exposed to 15-30μg/mL of ampicillin, and more than a 19-fold of increase in 150-300μg/mL of ampicillin exposure. DNA sequencing revealed that mpl and ampD were frequently mutated in these resistant strains. We performed a transcriptome analysis of deletion mutations of mpl or ampD, compared to PAO1. Both showed a two-fold increase in expression of quorum sensing (QS) genes including lasR and rhlI/R; the heightened expression was positively correlated with the expression of the ampicillin resistance gene ampC. We queried if quorum sensing contributes to the increase in the ampicillin MIC. After adding the quorum quencher acylase I, the growth yield both decreased by roughly 50% for Δmpl in 2000μg/mL of ampicillin and ΔampD in 4000μg/mL of ampicillin. Addition of the QS signals into synthase mutants restored the higher MIC, but only for the rhlI/R circuit. This study highlights the involvement of QS in antibiotic resistance evolution, and shows the multifactorial contributors to the observed phenotypes.

Selection of a Gentamicin-Resistant Variant Following Polyhexamethylene Biguanide (PHMB) Exposure in Escherichia coli Biofilms.

Antibiotic resistance is one of the most important issues facing modern medicine. Some biocides have demonstrated the potential of selecting resistance to antibiotics in bacteria, but data are still very scarce and it is important to better identify the molecules concerned and the underlying mechanisms. This study aimed to assess the potential of polyhexamethylene biguanide (PHMB), a widely used biocide in a variety of sectors, to select antibiotic resistance in Escherichia coli grown in biofilms. Biofilms were grown on inox coupons and then exposed daily to sublethal concentrations of PHMB over 10 days. Antibiotic-resistant variants were then isolated and characterized phenotypically and genotypically to identify the mechanisms of resistance. Repeated exposure to PHMB led to the selection of an E. coli variant (Ec04m1) with stable resistance to gentamycin (8-fold increase in minimum inhibitory concentration (MIC) compared to the parental strain. This was also associated with a significant decrease in the growth rate in the variant. Sequencing and comparison of the parental strain and Ec04m1 whole genomes revealed a nonsense mutation in the aceE gene in the variant. This gene encodes the pyruvate dehydrogenase E1 component of the pyruvate dehydrogenase (PDH) complex, which catalyzes the conversion of pyruvate to acetyl-CoA and CO2. A growth experiment in the presence of acetate confirmed the role of this mutation in a decreased susceptibility to both PHMB and gentamicin (GEN) in the variant. This work highlights the potential of PHMB to select resistance to antibiotics in bacteria, and that enzymes of central metabolic pathways should be considered as a potential target in adaptation strategies, leading to cross-resistance toward biocides and antibiotics in bacteria.

Fourier transform‐infrared assessment of cholesterol effect on antibiotic resistance of Salmonella Typhimurium

In this study, the effect of cholesterol on antibiotic resistance of Salmonella Typhimurium was investigated. Antibiotic resistance patterns revealed that the bacterium was susceptible to ampicillin, streptomycin, sulfamethoxazole/trimethoprim, ciprofloxacin, tetracycline, chloramphenicol, and gentamicin and resistant to penicillin, erythromycin, and vancomycin. The minimum inhibitory concentrations (MICs) of ampicillin, streptomycin, ciprofloxacin, and sulfamethoxazole/ trimethoprim antibiotics were found as 16, 128, 0.25, and 16 mg/L, respectively. When the effect of cholesterol on antibiotic resistivity was examined, it was found that MICs increased in cholesterol medium than in non-cholesterol medium. The increase in MICs and decrement in inhibition zones was considered as the decreasing effect of cholesterol on the sensitivity of Salmonella Typhimurium. The effect of cholesterol on the antibiotic sensitivity of Salmonella Typhimurium was also assessed by Fourier transformed infrared (FT-IR) spectroscopy, and the results indicated that new functional groups were observed at different wavelengths in the presence of cholesterol. Practical applications Antibiotic resistance is a global phenomenon resulting in the emergence of pathogens with resistance to clinically important antibiotics. Antibiotic resistance in foodborne pathogens such as Salmonella is a major concern for public health safety. Significant increase in bacterial resistance makes it increasingly necessary to develop new drugs and antibiotic activity modifiers and effective techniques for treatment. Fourier transformed infrared (FT-IR) can be used as a rapid and precise method for the assessment of cholesterol effect on antibiotic resistance pattern of Salmonella Typhimurium.

Mechanisms for Development of Ciprofloxacin Resistance in a Clinical Isolate of Pseudomonas aeruginosa.

Treatment of infections by Pseudomonas aeruginosa is difficult due to its high intrinsic and acquired antibiotic resistance. Upon colonization in the human hosts, P. aeruginosa accumulates genetic mutations that confer the bacterium antibiotic resistance and ability to better live in the host environment. Characterizing the evolutionary traits would provide important insights into the development of effective combinatory antibiotic therapies to cure P. aeruginosa infections. In this work, we performed a detailed analysis of the molecular mechanisms by which a clinical isolate (CSP18) yields a ciprofloxacin-resistant derivative (CRP42). Genomic DNA re-sequencing and RNAseq were carried out to compare the genomic mutational signature and transcriptional profiles between the two isolates. The results indicated that D87G mutation in GyrA, together with MexEF-OprN hyper-expression caused by F7S mutation in MexS, was responsible for the increased resistance to ciprofloxacin in the isolate CRP42. Further simulation of CRP42 by gene editing in CSP18 demonstrated that D87G mutation in GyrA rendered CSP18 a fourfold increase in minimum inhibitory concentration against ciprofloxacin, while F7S mutation in MexS conferred an additional eightfold increase. Our experimental results demonstrate for the first time that the clinically relevant F7S point mutation in MexS results in hyper-expression of the mexEF-oprN and thus confers P. aeruginosa resistance to ciprofloxacin.

1441. Using Carbapenem Resistance Levels to Discriminate Between Carbapenemase Producing and Non-Carbapenemase Producing Carbapenem Resistant Enterobacteriaceae

BackgroundCarbapenem-resistant Enterobacteriaceae (CRE) are a growing threat globally. Many CRE organisms carry plasmids that produce a carbapenemase enzyme (CP-CRE). Early detection of CP-CRE and aggressive infection prevention and control (IPC) measures are necessary to reduce transmission of resistant organisms and their plasmids. Many hospital laboratories do not test CRE for carbapenemase production. In Apr ‘18, the Philadelphia Department for Public Health (PDPH) established mandatory CRE reporting including isolate submission for mechanism testing at public health laboratories.MethodsWe analyzed trends of carbapenem resistance in CP and non-CP-CRE. We calculated sensitivity (Sen), specificity (Spe), positive and negative predictive values (PPV, NPV) at each level of carbapenem resistance to develop a predictive model with goal of effectively discriminating CP and non-CP CRE. ROC curves were plotted.ResultsFrom June,’18-Feb,’20, 351 CRE had genetic mechanism testing. 192 (54.7%) were Klebsiella pneumoniae, 53 (15.1%) were Enterobacter cloacae, 52 (14.8%) were E. coli, and the remaining 54 (15.4%) other Enterobacteriaceae.186 (53.0%) had a recorded minimum inhibitory concentration (MIC) for ertapenem, 191 (54.4%) for meropenem, 116 (33.0%) for imipenem, and 9 (2.6%) for doripenem. Doripenem was not further analyzed. The odds of being CP-CRE increased with increasing MIC. One standard dilution increase in MIC was associated with the following odds ratios (OR) of being CP-CRE: ertapenem OR 1.43 (95% CI: 1.11–1.83), imipenem OR 2.52 (95% CI: 1.7–3.793), meropenem, OR 2.20 (95% CI: 1.72–2.81). For each carbapenem, we calculated Sen, Spe, PPV, and NPV values for five MIC cut points; 0.5, 1, 2, 4, and 8 ug/ml, non-CP CRE defined as ≤ MIC cut point, CP-CRE defined as > cut point.Figure 1. ROC Curves Table 1. Sensitivity, Specificity, PPV, NPV of MIC Cut Points ConclusionThere were no MIC cut points that performed well in both Sen and Spe, however, for the purposes of IPC, correctly classifying CP CRE (Sen) is more important than correctly classifying non-CP CRE (Spe). MIC cut points that performed best were imipenem MIC of 1 ug/ml and meropenem MIC of 1 ug/ml, with Sen 98%, 94% respectively, PPV 85%, 84% respectively and NPV 88%, 76% respectively. When further testing is not available, MIC cut points may be used to infer CP production.Disclosures All Authors: No reported disclosures

In Vitro Activities and Inoculum Effects of Ceftazidime-Avibactam and Aztreonam-Avibactam against Carbapenem-Resistant Enterobacterales Isolates from South Korea

Ceftazidime-avibactam (CAZ-AVI) and aztreonam-avibactam (AZT-AVI) are novel antibiotic combinations active against multidrug-resistant Gram-negative pathogens. This study aimed to evaluate their in vitro activities and inoculum effects in carbapenem-resistant Enterobacterales (CRE), including carbapenemase-producing (CP)-CRE and non-CP-CRE. A total of 81 independent clinical isolates of carbapenem-resistant Escherichia coli and Klebsiella pneumoniae were collected. CAZ-AVI and AZT-AVI minimal inhibitory concentrations (MICs) were evaluated by broth microdilution using standard and high inocula. The inoculum effect was defined as an ≥8-fold increase in MIC with high inoculum. Phenotypic determination of β-lactam resistance mechanism and PCR for carbapenemase genes were performed. Of the 81 CRE isolates, 35 (43%) were CP-CRE. Overall, 73% of the isolates were susceptible to CAZ-AVI, and 95% had low AZT-AVI MICs (≤8 µg/mL). The MIC50/MIC90s of CAZ-AVI and AZT-AVI were 4/≥512 µg/mL and 0.5/4 µg/mL, respectively. CAZ-AVI was more active against non-CP-CRE than against CP-CRE (susceptibility 80% vs. 63%, p = 0.08; MIC50/MIC90, 2/16 μg/mL vs. 4/≥512 μg/mL), whereas AZT-AVI was more active against CP-CRE (MIC50/MIC90, 0.25/1 μg/mL vs. 0.5/8 μg/mL). All four isolates with high AZT-AVI MIC (≥16 μg/mL) were resistant to CAZ-AVI, but only 18% (4/22) of CAZ-AVI-resistant isolates had high AZT-AVI MIC. The rates of the inoculum effect for CAZ-AVI and AZT-AVI were 18% and 47%, respectively (p < 0.001). Interestingly, the frequency of the AZT-AVI inoculum effect was higher in K. pneumoniae than E. coli (64% vs. 8%, p < 0.001). AZT-AVI is more active against CRE than CAZ-AVI, even in CP-CRE and CAZ-AVI-resistant isolates. The presence of a substantial inoculum effect may contribute to clinical failure in high-inoculum infections treated with AZT-AVI.