Characterisation of a novel AmpC beta-lactamase, DHA-33, resistant to inhibition by cloxacillin.

Plasmid-encoded DHA-type AmpCs have been extensively reported in Enterobacterales. The expression of the genes encoding these plasmid-mediated enzymes are inducible and these enzymes are capable of conferring resistance to a wide spectrum of beta-lactams including penicillins and broad-spectrum cephalosporins. The identification of infections caused by AmpC-producing bacteria is a necessity, both for infection control/epidemiology purposes and to inform treatment choices. A common testing method for AmpC production in the clinical laboratory setting is to supplement Mueller-Hinton agar plates used for antibiotic disk diffusion with cloxacillin, a potent inhibitor of AmpC enzymes. Here we describe a novel DHA variant, produced by a clinical Escherichia coli isolate, which is resistant to cloxacillin inhibition.

Negatively controlled trial investigating the effects of dry cow therapy on clinical mastitis and culling in multiparous cows.

Blanket dry cow therapy (DCT) is a major contributor to overall antibiotic usage on dairy farms in the United States. With low prevalence of intramammary infections at dry-off in US herds today, alternative DCT approaches have been the focus of much research. We hypothesized that complete cessation of DCT [i.e., use of internal teat sealants (ITS) only at dry-off] could be a practical alternative to blanket DCT in well-managed herds. The objective of this negatively controlled clinical trial was to determine the effects of DCT on clinical mastitis (CM) and removal from the herd during the dry period and the first 200 d of the subsequent lactation in multiparous dairy cows treated with only ITS at dry-off. As a secondary objective, we conducted exploratory analysis to identify subpopulations in the herd (based on parity, previous CM history, and dry-period length) where DCT would not affect postcalving udder health, to generate hypotheses about potential alternative selective DCT programs. The study was conducted in a commercial dairy herd in South Dakota from June 2020 to January 2021. Dry-off sessions (n = 43) were scheduled such that all cows at a given session were dried off using ITS alone (ITS only, n = 20 sessions, n = 1,108 cows) or an intramammary DCT product containing 500 mg of cloxacillin (Dry-Clox, Boehringer Ingelheim) followed by ITS (ITS+ABX, n = 23 sessions, n = 1,331 cows). Culling and CM events were recorded by farm workers who were blinded to the treatment status of cows. Hazard ratios (HR) for the effects of the treatment group on CM and removal from the herd were estimated using multivariable Cox proportional hazards, adjusting for the clustered treatment allocation strategy. Risk of removal from the herd during the dry period was lower in ITS+ABX than ITS-only cows (1.1 vs. 2.7%; HR = 0.45; 95% CI: 0.25 to 0.81). Risk of removal from the herd during the first 200 d of lactation was similar in ITS+ABX and ITS-only cows (17.3 vs. 18.0%; HR = 0.98; 95% CI: 0.82 to 1.18). Risk of CM during the first 200 d of lactation was lower in ITS+ABX cows (6.9%; HR = 0.56; 95% CI: 0.41 to 0.76) compared with ITS-only cows (13.4%). The beneficial effects of DCT on CM and removal from the herd were consistently observed across strata of parity, previous CM history, and dry-period length, indicating that no subpopulations could be identified to withhold DCT. The findings from this study indicate that the omission of DCT from the dry-off procedure, when udder health is not taken into consideration, in multiparous cows can have a negative effect on cow health and welfare. Findings from previous research suggest that culture- or algorithm-guided selective dry cow therapy are likely to be safer approaches to improving antibiotic stewardship.

Synergistic Combinations of FDA-Approved Drugs with Ceftobiprole against Methicillin-Resistant Staphylococcus aureus.

New strategies are urgently needed to address the public health threat of antimicrobial resistance. Synergistic agent combinations provide one possible pathway toward addressing this need and are also of fundamental mechanistic interest. Effective methods for comprehensively identifying synergistic agent combinations are required for such efforts. In this study, an FDA-approved drug library was screened against methicillin-resistant Staphylococcus aureus (MRSA) (ATCC 43300) in the absence and presence of sub-MIC levels of ceftobiprole, a PBP2a-targeted anti-MRSA ß-lactam. This screening identified numerous potential synergistic agent combinations, which were then confirmed and characterized for synergy using checkerboard analyses. The initial group of synergistic agents (sum of the minimum fractional inhibitory concentration ∑FICmin ≤0.5) were all ß-lactamase-resistant ß-lactams (cloxacillin, dicloxacillin, flucloxacillin, oxacillin, nafcillin, and cefotaxime). Cloxacillin-the agent with the greatest synergy with ceftobiprole-is also highly synergistic with ceftaroline, another PBP2a-targeted ß-lactam. Further follow-up studies revealed a range of ceftobiprole synergies with other ß-lactams, including with imipenem, meropenem, piperacillin, tazobactam, and cefoxitin. Interestingly, given that essentially all other ceftobiprole-ß-lactam combinations showed synergy, ceftaroline and ceftobiprole showed no synergy. Modest to no synergy (0.5 < ∑FICmin ≤ 1.0) was observed for several non-ß-lactam agents, including vancomycin, daptomycin, balofloxacin, and floxuridine. Mupirocin had antagonistic activity with ceftobiprole. Flucloxacillin appeared particularly promising, with both a low intrinsic MIC and good synergy with ceftobiprole. That so many ß-lactam combinations with ceftobiprole show synergy suggests that ß-lactam combinations can generally increase ß-lactam effectiveness and may also be useful in reducing resistance emergence and spread in MRSA. IMPORTANCE Antimicrobial resistance represents a serious threat to public health. Antibacterial agent combinations provide a potential approach to combating this problem, and synergistic agent combinations-in which each agent enhances the antimicrobial activity of the other-are particularly valuable in this regard. Ceftobiprole is a late-generation ß-lactam antibiotic developed for MRSA infections. Resistance has emerged to ceftobiprole, jeopardizing this agent's effectiveness. To identify synergistic agent combinations with ceftobiprole, an FDA-approved drug library was screened for potential synergistic combinations with ceftobiprole. This screening and follow-up studies identified numerous ß-lactams with ceftobiprole synergy.

Frequency of drug-induced liver injury in children receiving anti-staphylococcal penicillins.

INTRODUCTION: Anti-staphylococcal penicillins (ASPs) are among the most commonly prescribed antibiotics in children and are associated with a risk of drug-induced liver injury (DILI). Despite the frequent use of ASPs in children, there is no consensus on whether liver function tests (LFTs) should be routinely monitored during treatment. OBJECTIVES: To review the literature on the frequency of ASP-related DILI in children to determine the incidence, risk factors and outcomes of hepatotoxicity. METHODS: PubMed, MEDLINE and Embase were searched in January 2022 for original studies of children who received cloxacillin, dicloxacillin, flucloxacillin, methicillin, nafcillin or oxacillin that included ≥10 children aged up to 18 years, and presented data on the incidence of DILI in children exposed to ASPs. RESULTS: Overall, two studies of oral flucloxacillin, two of intravenous (IV) methicillin, three of IV nafcillin and four of IV oxacillin were included. The mean onset of DILI ranged between 7.0 and 19.0 days following commencement of antibiotic treatment and all episodes resolved between 14.2 and 16.0 days after drug discontinuation, with no specific treatment required. This review found that the incidence of DILI in children was 1 in 50 000 for oral flucloxacillin and ranged from 1 in 3 to 13 for IV oxacillin, methicillin and nafcillin. CONCLUSIONS: This review found that routine LFT monitoring is not required in children receiving low dose oral flucloxacillin in a primary care setting, although pharmacovigilance is critical. For IV preparations, the existing data support routine LFT monitoring in those receiving treatment for at least 7 days.

Pharmacokinetic-Pharmacodynamic Study of Ampicillin-Cloxacillin Combination in Indian Thoroughbred Horses (Equus caballus) and Safety Evaluation of the Computed Dosage Regimen.

The pharmacokinetics of ampicillin-cloxacillin, given as single intravenously dose of 10 mg.kg-1 (5 mg.kg-1 of ampicillin plus 5 mg.kg-1 of cloxacillin) was examined in clinically presented Indian thoroughbred horses (n = 6) in order to design appropriate dosing strategies. Drug concentrations in plasma were determined by high performance liquid chromatography (HPLC) and pharmacokinetic parameters were derived by non-compartmental analysis using WinNonlin software. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of ampicillin-cloxacillin against quality control strains of Escherichia coli and Staphylococcus aureus, grown in Muller Hinton Broth, were determined by broth microdilution method. For ampicillin, area under plasma drug concentration time curve (AUC) was 15.2 ± 0.54 µg.h.ml-1, mean residence time (MRT) was 1.33 ± 0.06 h and clearance (Cl) was 0.33 ± 0.01 L.h-1.kg-1. For cloxacillin, AUC was 18.0 ± 0.9 µg.h.ml-1, MRT was 1.28 ± 0.02 h and Cl was 0.28 ± 0.01 L.h-1.kg-1. MIC of ampicillin-cloxacillin combination against E. coli and S. aureus was determined to be 0.4 µg.ml-1. PK-PD integration indicated that to maintain %T > MIC value 50% for bacteria with MIC ≤ 0.4 µg.ml-1, an appropriate intravenous dosage regimen of ampicillin-cloxacillin combination in horses would be 15 mg.kg-1 (i.e. 7.5 mg.kg-1 of ampicillin plus 7.5 mg.kg-1 of cloxacillin), to be repeated at 12 h intervals. Safety profile of the recommended regimen did not significantly alter any of the 16 biochemical or haematological parameters studied.

Empirical use of ß-lactam/ß-lactamase inhibitor combinations does not increase mortality compared with cloxacillin and cefazolin in methicillin-susceptible Staphylococcus aureus bacteraemia: a propensity-weighted cohort study.

OBJECTIVES: To evaluate the effectiveness of empirical therapy with ß-lactam/ß-lactamase inhibitor combinations (BL/BLICs) for MSSA bacteraemia. METHODS: We conducted a post hoc analysis of all adult patients with MSSA bacteraemia who were hospitalized at a Spanish university hospital between 2013 and 2018. We compared 30 day mortality among patients receiving initial therapy with BL/BLICs (de-escalated to cloxacillin or cefazolin within 96 h) versus cloxacillin or cefazolin, using propensity score analysis with the inverse probability of treatment weighting (IPTW) method. RESULTS: We evaluated 373 patients with MSSA bacteraemia. Among them, 198 patients met the eligibility criteria, including 127 patients in the BL/BLICs group and 71 patients in the cloxacillin/cefazolin group. Patients in the BL/BLICs group had a higher Charlson comorbidity index (median, 2 [IQR, 1-4.5] versus 2 [IQR, 0-4]); an increased proportion of high-risk sources (i.e. endocarditis, respiratory sources and bacteraemia of unknown origin [34.6% versus 18.3%]); and an earlier start of antibiotic treatment (median, 0 days [IQR, 0-0] versus 1 day [IQR, 1-2]). Thirty day mortality did not significantly differ between the BL/BLICs and the cloxacillin/cefazolin groups (27 patients [21.3%] versus 13 patients [18.3%]; IPTW-adjusted OR = 0.53 [95% CI, 0.18-1.51]). For secondary outcomes, 7 day mortality and 90 day relapse were not statistically different between study groups (8.7% versus 5.6% [P = 0.62] and 6.2% versus 3.8% [P = 0.81], respectively). CONCLUSIONS: BL/BLICs might be an effective empirical treatment for MSSA bacteraemia when de-escalated to cloxacillin or cefazolin within 96 h from the index blood culture.

Synergistic Activity of Lupinifolin in Combinations with Antibiotics Against Staphylococcus aureus.

<b>Background and Objective:</b> Antibacterial resistance is one of the top global public health problems. The use of natural substances, which can enhance the antibacterial activity of currently used medications, is a promising alternative to oppose antibacterial resistance. The pharmacological activities of lupinifolin, a prenylated flavanone isolated from stems of <i>Derris reticulata</i> Craib., against growth and biofilm formation of <i>Streptococcus mutans</i> and <i>Staphylococcus aureus</i> have been previously documented. Nonetheless, interactions between lupinifolin and other antibacterial agents have not been determined. This study aimed to investigate the effects of lupinifolin in combinations with some antibacterial agents, specifically ampicillin, cloxacillin or vancomycin, against <i>S. mutans</i>, Methicillin-Sensitive <i>S. aureus</i> (MSSA) and Methicillin-Resistant <i>S. aureus</i> (MRSA). <b>Materials and Methods:</b> The checkerboard assay was performed to determine the antibacterial activity of lupinifolin plus the testing antibacterial agents. The Fractional Inhibitory Concentration Index (FICI) was calculated to indicate the interaction between lupinifolin and the antibacterial agent tested. <b>Results:</b> Lupinifolin exerted the synergistic activity when using in combination with ampicillin or cloxacillin against MSSA with the FICIs of <u><</u>0.5. The potential synergistic effect was also observed with lupinifolin plus ampicillin or cloxacillin against MRSA. However, the combination of lupinifolin plus vancomycin resulted in no interaction against MRSA. The combined effects of lupinifolin and ampicillin or cloxacillin against <i>S. mutans</i> were somewhat ambiguous with the borderline values of FICI of 0.5156 and 0.5625, respectively. <b>Conclusion:</b> Lupinifolin potentially plays a role as an antibacterial intensifier against some pathogenic gram-positive bacteria, particularly MSSA and MRSA. Nonetheless, further experiments are required to explain the precise mechanism of synergy.

Osseointegration and antibacterial effect of an antimicrobial peptide releasing mesoporous titania implant.

Medical devices such as orthopedic and dental implants may get infected by bacteria, which results in treatment using antibiotics. Since antibiotic resistance is increasing in society there is a need of finding alternative strategies for infection control. One potential strategy is the use of antimicrobial peptides, AMPs. In this study, we investigated the antibiofilm effect of the AMP, RRP9W4N, using a local drug-delivery system based on mesoporous titania covered titanium implants. Biofilm formation was studied in vitro using a safranine biofilm assay and LIVE/DEAD staining. Moreover, we investigated what effect the AMP had on osseointegration of commercially available titanium implants in vivo, using a rabbit tibia model. The results showed a sustained release of AMP with equal or even better antibiofilm properties than the traditionally used antibiotic Cloxacillin. In addition, no negative effects on osseointegration in vivo was observed. These combined results demonstrate the potential of using mesoporous titania as an AMP delivery system and the potential use of the AMP RRP9W4N for infection control of osseointegrating implants.

Detection of various beta-Lactamases in Escherichia coli and Klebsiella sp.: A study from Tertiary Care Centre of North India.

Objective: The emergence of carbapenem-resistant Escherichia coli and Klebsiella species is a global threat. We aimed to compare two phenotypic methods and evaluate the genotypic method for the detection of beta-lactamases produced by E. coli and Klebsiella spp. Materials and Methods: One hundred and twenty-six E. coli and Klebsiella isolates were examined for phenotypic production of beta-lactamases by using disc diffusion, combined disc test (CDT) and modified carbapenem inactivation method (mCIM). All strains were also studied for the presence of various genes by polymerase chain reaction. Results: Out of 126 isolates, 96% of the isolates were extended-spectrum ß-lactamase (ESBL) producers based on the presence of various ESBL genes. CDT method showed higher number of total (89%) carbapenemases in comparison to mCIM (81%). Among carbapenemases none of the isolates were Klebsiella pneumoniae carbapenemase producer by CDT, while 69% isolates were metallo-beta-lactamase (MBL) producers. Another method, mCIM/ethylene diamine tetraacetic acid mCIM showed 100% agreement for MBL detection. As regards, AmpC and class D carbapenemases; 0.04% and 16% positivity was detected, respectively, based on CDT method. Molecular analysis revealed 91% of the isolates harbouring carbapenemase genes. blaNDMwas the most common gene detected followed byblaOXA-48. Nine of the blaNDM-positive isolates also possessed blaOXA-48gene. Conclusion: Our finding shows high percentages of ESBL and carbapenemases in E. coli and Klebsiella spp. Among phenotypic methods, CDT seems to be a better choice as prevalence of carbapenemases shows lots of variation in our country. For Class B enzymes, both CDT and mCIM/eCIM can be used in the routine laboratories.

Conjugative plasmidic AmpC detected in Escherichia coli, Proteus mirabilis and Klebsiella pneumoniae human clinical isolates from Portugal.

AmpC is a type of ß-lactamase enzyme produced by bacteria; these enzymes are classified in Class C and Group 1, and these confer resistance to cephamycin. Enterobacterales producing AmpC are reported worldwide and have great clinical importance due to therapeutic restriction and epidemiological importance once the easy dissemination by plasmidic genes to other bacteria is a real threat. These genes are naturally found in some enterobacteria as Enterobacter cloacae, Morganella morganii, and Citrobacter freundii, but other species have demonstrated similar resistance phenotype of AmpC production. Genes carried in plasmids have been described in these species conferring resistance to cefoxitin and causing therapeutic failure in some bacterial infections. This work detected and described five clinical strains of Escherichia coli, Proteus mirabilis, and Klebsiella pneumoniae that presented plasmid ampC (pAmpC) isolated from the north of Portugal collected in 2009. AmpC production was confirmed by inhibition of the enzyme by cloxacillin and boronic acid in agar diffusion tests. Also, PCR (polymerase chain reaction) was performed for the detection of gene universal to AmpC, blaampC, and others to AmpC group: blaACC, blaCIT, blaCMY, blaDHA, and blaEBC. The conjugation in liquid medium for 24 h was realized to determine if gene is localized in chromosome or plasmid. The isolates and their conjugants showed phenotypic characteristics and blaCMY and blaCIT were detected by PCR corroborating the AmpC characteristics observed in these bacteria. Confirmation of transfer of plasmid containing genes encoding AmpC is of high epidemiological relevance to the hospital studied and demonstrated the importance of AmpC surveillance and studies in hospital and community environments in order to choose the appropriate therapy for bacterial infections.

Do Antibiotics Potentiate Proteases in Hemotoxic Snake Venoms?

Antibiotics are often administered with antivenom following snakebite envenomings in order to avoid secondary bacterial infections. However, to this date, no studies have evaluated whether antibiotics may have undesirable potentiating effects on snake venom. Herein, we demonstrate that four commonly used antibiotics affect the enzymatic activities of proteolytic snake venom toxins in two different in vitro assays. Similar findings in vivo could have clinical implications for snakebite management and require further examination.

In vitro Reducing Effect of Cloxacillin on Minimum Inhibitory Concentrations to Imipenem, Meropenem, Ceftazidime, and Cefepime in Carbapenem-resistant Pseudomonas aeruginosa Isolates.

Today, resistance to antibacterial agents is the most important problem facing public health. Pseudomonas aeruginosa is a common gram-negative bacterium and an important cause of nosocomial infections. Resistance to many antibiotics in strains of P. aeruginosa isolated from hospital settings such as cephalosporins and carbapenems have been recently reported. Therefore, the introduction of a new strategy to treat the infection of these organisms will be beneficial. In this study we determined the ability of cloxacillin to reduce Minimum Inhibitory Concentrations (MICs) of carbapenem-resistant P. aeruginosa to imipenem (IMI), meropenem (MEM), ceftazidime (CAZ), and cefepime (FEP). From 2015 to 2017, 61 non-duplicates of carbapenem-resistant P. aeruginosa were collected from clinical samples of hospitalized patients in Kerman, Iran. The MICs of the isolates to IMI, MEM, CAZ, and FEP with/without cloxacillin were determined by microbroth dilution method. The level of MIC of isolates to carbapenems (IMI and MEM) and cephalosporins (CAZ and FEP) ranged from 1-256 µg/mL and 4-1024 µg/mL alone and from 1-32 µg/mL and 1-512 µg/mL in combination with cloxacillin, respectively. The MIC showed a significant difference reduction after the addition of cloxacillin (P ≤ 0.05). Our results showed in vitro potentially of cloxacillin in reduction of MIC to IMI, MEM, CAZ, and FEP in multi-drug resistant P. aeruginosa, therefore combination of these antibiotics with cloxacillin could be beneficial for treatment of infections caused by multi-drug resistant P. aeruginosa.

Differential interactions of the ß-lactam cloxacillin with human renal organic anion transporters (OATs).

The ß-lactam penicillin antibiotic cloxacillin (CLX) presents wide inter-individual pharmacokinetics variability. To better understand its molecular basis, the precise identification of the detoxifying actors involved in CLX disposition and elimination would be useful, notably with respect to renal secretion known to play a notable role in CLX elimination. The present study was consequently designed to analyze the interactions of CLX with the solute carrier transporters organic anion transporter (OAT) 1 and OAT3, implicated in tubular secretion through mediating drug entry at the basolateral pole of renal proximal cells. CLX was first shown to block OAT1 and OAT3 activity in cultured OAT-overexpressing HEK293 cells. Half maximal inhibitory concentration (IC50 ) value for OAT3 (13 µm) was however much lower than that for OAT1 (560 µm); clinical inhibition of OAT activity and drug-drug interactions may consequently be predicted for OAT3, but not OAT1. OAT3, unlike OAT1, was next shown to mediate CLX uptake in OAT-overexpressing HEK293 cells. Kinetic parameters for this OAT3-mediated transport of CLX (Km  = 10.7 µm) were consistent with a possible in vivo saturation of this process for high CLX plasma concentrations. OAT3 is consequently likely to play a pivotal role in renal CLX secretion and consequently in total renal CLX elimination, owing to the low plasma unbound fraction of the antibiotic. OAT3 genetic polymorphisms as well as co-administered drugs inhibiting in vivo OAT3 activity may therefore be considered as potential sources of CLX pharmacokinetics variability.

Short communication: Investigation of extra-intestinal pathogenic Escherichia coli virulence genes, bacterial motility, and multidrug resistance pattern of strains isolated from dairy cows with different severity scores of clinical mastitis.

Escherichia coli is a major pathogen involved in the etiology of environmentally derived bovine mastitis and is characterized by a variety of virulence factors (VF). Mammary infections with E. coli have shown a wide range of clinical signs, causing changes in milk (score 1, or mild), abnormal appearance of milk and udder inflammation (score 2, or moderate), and abnormalities in milk, udder inflammation, and systemic signs of illness (score 3, or severe). Nevertheless, to date, the profile of the genes related to the virulence of the pathogen in mammary infections and the severity scores of cases have not been thoroughly elucidated. Therefore, a panel of 18 virulence-encoding genes associated with extra-enteric pathogenicity of E. coli (ExPEC) were investigated in addition to in vitro swimming and swarming motility profiles and antimicrobial susceptibility/resistance patterns among 114 E. coli strains isolated from cows with clinical mastitis and different severity scores. Of 114 clinical cases, 39.5, 54.4, and 6.1% were mild, moderate, and severe, respectively. The main genes related to VF harbored by isolates were adhesins (fimH 100%; ecpA 64.0%, fimA 31.6%), serum resistance (traT 81.6%; ompT 35.1%), siderophores (irp2 9.6%), and hemolysin (hlyA 7%). Among the isolates studied, 99.1% showed in vitro resistance to bacitracin and cloxacillin, and 98.2% to lincosamin. Of the total isolates, 98.2% were considered multidrug resistant based on the multiple antimicrobial resistance index. No significant difference was observed between mean swimming (13.8 mm) and swarming (13.5 mm) motility, as well as severity scores of clinical mastitis and the ExPEC genes studied. The isolation of strains resistant to various antimicrobials, even though tested only in vitro, highlights the importance of rational use of antimicrobials for mastitis treatment. The high prevalence of the genes related to serum resistance (traT and ompT) and adhesion (ecpA) of the pathogen, in addition to main associations between the genes fimH, ecpA, and traT among cows with severity scores of 1 (15%) and 2 (22.6%), indicates that the genes traT, ecpA, and ompT could be further studied as biomarkers of ExPEC for clinical intramammary infections. In addition, the ExPEC genes ompT (protectin), ibe10 (invasin), and ecpA (adhesin) were investigated for the first time among cows with mastitis, where scores of clinical severity were assessed. Results of this study contribute to the characterization of virulence mechanisms and antimicrobial resistance profile of ExPEC variants that affect dairy cows with different scores of clinical mastitis.

Inhibition of Drug Resistance of Staphylococcus aureus by Efflux Pump Inhibitor and Autolysis Inducer to Strengthen the Antibacterial Activity of ß-lactam Drugs.

This study explored a potential treatment against methicillin-resistant Staphylococcus aureus (MRSA) infections that combines thioridazine (TZ), an efflux pump inhibitor, and miconazole (MCZ), an autolysis inducer, with the anti-microbial drug cloxacillin (CXN). In vitro, the combination treatment of TZ and MCZ significantly reduced 4096-fold (Σ (FIC) = 0.1 - 1.25) the MIC value of CXN against S. aureus. In vivo, the combination therapy significantly relieved breast redness and swelling in mice infected with either clinical or standard strains of S. aureus. Meanwhile, the number of bacteria isolated from the MRSA135-infected mice decreased significantly (p = 0.0427 < 0.05) after the combination therapy when compared to monotherapy. Moreover, the number of bacteria isolated from the mice infected with a reference S. aureus strain also decreased significantly (p = 0.0191 < 0.05) after the combination therapy when compared to monotherapy. The pathological changes were more significant in the CXN-treated group when compared to mice treated with a combination of three drugs. In addition, we found that combination therapy reduced the release of the bacteria-stimulated cytokines such as IL-6, IFN-γ, and TNF-α. Cytokine assays in serum revealed that CXN alone induced IL-6, IFN-γ, and TNF-α in the mouse groups infected with ATCC 29213 or MRSA135, and the combination of these three drugs significantly reduced IL-6, IFN-γ, and TNF-α concentrations. Also, the levels of TNF-α and IFN-γ in mice treated with a combination of three drugs were significantly lower than in the CXN-treated group. Given the synergistic antibacterial activity of CXN, we concluded that the combination of CXN with TZ, and MCZ could be developed as a novel therapeutic strategy against S. aureus.This study explored a potential treatment against methicillin-resistant Staphylococcus aureus (MRSA) infections that combines thioridazine (TZ), an efflux pump inhibitor, and miconazole (MCZ), an autolysis inducer, with the anti-microbial drug cloxacillin (CXN). In vitro, the combination treatment of TZ and MCZ significantly reduced 4096-fold (Σ (FIC) = 0.1 ­ 1.25) the MIC value of CXN against S. aureus. In vivo, the combination therapy significantly relieved breast redness and swelling in mice infected with either clinical or standard strains of S. aureus. Meanwhile, the number of bacteria isolated from the MRSA135-infected mice decreased significantly (p = 0.0427 < 0.05) after the combination therapy when compared to monotherapy. Moreover, the number of bacteria isolated from the mice infected with a reference S. aureus strain also decreased significantly (p = 0.0191 < 0.05) after the combination therapy when compared to monotherapy. The pathological changes were more significant in the CXN-treated group when compared to mice treated with a combination of three drugs. In addition, we found that combination therapy reduced the release of the bacteria-stimulated cytokines such as IL-6, IFN-γ, and TNF-α. Cytokine assays in serum revealed that CXN alone induced IL-6, IFN-γ, and TNF-α in the mouse groups infected with ATCC 29213 or MRSA135, and the combination of these three drugs significantly reduced IL-6, IFN-γ, and TNF-α concentrations. Also, the levels of TNF-α and IFN-γ in mice treated with a combination of three drugs were significantly lower than in the CXN-treated group. Given the synergistic antibacterial activity of CXN, we concluded that the combination of CXN with TZ, and MCZ could be developed as a novel therapeutic strategy against S. aureus.

Phenotypic and genetic resistance traits of Pseudomonas aeruginosa strains infecting cystic fibrosis patients: A French cohort study.

Pseudomonas aeruginosa is responsible for chronic respiratory tract colonisation and acute exacerbations in cystic fibrosis (CF) patients. This Gram-negative bacterium often develops multidrug resistance, which represents a therapeutic challenge. The objective of this study was to characterise the phenotypic and genetic ß-lactam resistance traits of P. aeruginosa strains isolated from CF patients at Grenoble Alpes University Hospital (Grenoble, France). The susceptibility to ß-lactam compounds of 123 P. aeruginosa strains collected from the lower respiratory tract of 45 CF patients between 2010-2014 was evaluated. Genetic analyses focused on characterisation of the presence of carbapenemase- and extended-spectrum ß-lactamases (ESBL)-encoding genes as well as alterations in the oprD gene encoding the OprD porin. Among the 123 P. aeruginosa strains evaluated, 25 were susceptible to both ceftazidime (CAZ) and imipenem (IPM), 9 only to IPM and 36 only to CAZ; 53 strains were resistant to both drugs. CAZ resistance could be reverted by cloxacillin in 29 strains, indicating overproduction of cephalosporinase. Genetic analyses performed for 79 P. aeruginosa strains revealed no ESBL- or carbapenemases-encoding genes. Among the 74 IPM-resistant strains, 42 (56.8%) displayed major alterations in the OprD protein sequence. This study shows that in this CF patient cohort, cephalosporinase overproduction and OprD alterations were the main resistance mechanisms of P. aeruginosa to CAZ and IPM, respectively. No genes coding for ESBLs or carbapenemases were detected, but monitoring of the emergence of such resistance genes in CF patients is warranted owing to their ability to rapidly spread by horizontal gene transfer.

Chitosan and cloxacillin combination improve antibiotic efficacy against different lifestyle of coagulase-negative Staphylococcus isolates from chronic bovine mastitis.

Bovine mastitis affects the health of dairy cows and the profitability of herds worldwide. Coagulase-negative staphylococci (CNS) are the most frequently isolated pathogens in bovine intramammary infection. Based on the wide range of antimicrobial, mucoadhesive and immunostimulant properties demonstrated by chitosan, we have evaluated therapy efficiency of chitosan incorporation to cloxacillin antibiotic as well as its effect against different bacterial lifestyles of seven CNS isolates from chronic intramammary infections. The therapeutic effects of combinations were evaluated on planktonic cultures, bacterial biofilms and intracellular growth in mammary epithelial cells. We found that biofilms and intracellular growth forms offered a strong protection against antibiotic therapy. On the other hand, we found that chitosan addition to cloxacillin efficiently reduced the antibiotic concentration necessary for bacterial killing in different lifestyle. Remarkably, the combined treatment was not only able to inhibit bacterial biofilm establishment and increase preformed biofilm eradication, but it also reduced intracellular bacterial viability while it increased IL-6 secretion by infected epithelial cells. These findings provide a new approach to prophylactic drying therapy that could help to improve conventional antimicrobial treatment against different forms of bacterial growth in an efficient, safer and greener manner reducing multiresistant bacteria generation and spread.

Boesenbergia rotunda (L.) Mansf. extract potentiates the antibacterial activity of some ß-lactams against ß-lactam-resistant staphylococci.

OBJECTIVES: The purpose of this study was to investigate the effect of Boesenbergia rotunda (L.) Mansf. extract (BRE) and peptidoglycan inhibitor antibiotics, alone and in combination, against ß-lactam-resistant staphylococci. METHODS: Antibacterial and synergistic activities of BRE alone and in combination with ampicillin (AMP), cloxacillin (CLX), cefazolin (CZO) or vancomycin (VAN) were evaluated against two ß-lactam-resistant Staphylococcus aureus (BRSA) isolates and one ß-lactam-resistant Staphylococcus epidermidis (BRSE) isolate. The activities were confirmed by killing curve assays. The preliminary antimicrobial action was elucidated by transmission electron microscopy (TEM) and cytoplasmic membrane (CM) permeability assay. RESULTS: All tested staphylococci were inhibited by BRE at a minimum inhibitory concentration (MIC) of 16µg/mL. Two BRSA strains showed high resistance to CLX, AMP and CZO, whilst BRSE was resistant to CLX and AMP. All tested isolates remained susceptible to VAN. Chequerboard assay demonstrated a fractional inhibitory concentration index (FICI) of 0.50 for the BRE+CLX combination against the BRSA strains. Killing curve determinations confirmed the antibacterial and synergistic activities. TEM revealed collapse of the CM in BRE-treated cells and damage both of the CM and peptidoglycan (PG) in BRE+CLX-treated cells. The CM permeability assay showed that either BRE or nisin alone as well as BRE+CLX significantly induced leakage of OD260nm-absorbing materials. CONCLUSIONS: BRE potentiated the activity of ß-lactams, particularly CLX, against ß-lactam-resistant staphylococci by damaging the CM and PG layer, leading to leakage of intracellular material. Combination of BRE and ß-lactams provides a potential way forward in developing novel antistaphylococcal agents.

The performance of a resazurin chromogenic agar plate with a combined disc method for rapid screening of extended-spectrum-ß-lactamases, AmpC ß-lactamases and co-ß-lactamases in Enterobacteriaceae.

A promising means of rapid screening of extended-spectrum-ß-lactamase (ESBL), AmpC ß-lactamase, and co-production of ESBL and AmpC that combines resazurin chromogenic agar (RCA) with a combined disc method is here reported. Cefpodoxime (CPD) discs with and without clavulanic acid (CA), cloxacillin (CX) and CA+CX were evaluated against 86 molecularly confirmed ß-lactamase-producing Enterobacteriaceae, including 15 ESBLs, 32 AmpCs, nine co-producers of ESBL and AmpC and 30 carbapenemase producers. The CA and CX synergy test successfully detected all ESBL producers (100% sensitivity and 98.6% specificity) and all AmpC producers (100% sensitivity and 96.36% specificity). This assay also performed well in screening for co-existence of ESBL and AmpC (88.89% sensitivity and 100% specificity). The RCA assay is simple and inexpensive and provides results within 7 hr. It can be performed in any microbiological laboratory, in particular, in geographic regions in which ESBL, AmpC or co-ß-lactamase-producing Enterobacteriaceae are endemic.

Removal of antibiotic cloxacillin by means of electrochemical oxidation, TiO2 photocatalysis, and photo-Fenton processes: analysis of degradation pathways and effect of the water matrix on the elimination of antimicrobial activity.

This study evaluates the treatment of the antibiotic cloxacillin (CLX) in water by means of electrochemical oxidation, TiO2 photocatalysis, and the photo-Fenton system. The three treatments completely removed cloxacillin and eliminated the residual antimicrobial activity from synthetic pharmaceutical wastewater containing the antibiotic, commercial excipients, and inorganic ions. However, significant differences in the degradation routes were found. In the photo-Fenton process, the hydroxyl radical was involved in the antibiotic removal, while in the TiO2 photocatalysis process, the action of both the holes and the adsorbed hydroxyl radicals degraded the pollutant. In the electrochemical treatment (using a Ti/IrO2 anode in sodium chloride as supporting electrolyte), oxidation via HClO played the main role in the removal of CLX. The analysis of initial by-products showed five different mechanistic pathways: oxidation of the thioether group, opening of the central ß-lactam ring, breakdown of the secondary amide, hydroxylation of the aromatic ring, and decarboxylation. All the oxidation processes exhibited the three first pathways. Moreover, the aromatic ring hydroxylation was found in both photochemical treatments, while the decarboxylation of the pollutant was only observed in the TiO2 photocatalysis process. As a consequence of the degradation routes and mechanistic pathways, the elimination of organic carbon was different. After 480 and 240 min, the TiO2 photocatalysis and photo-Fenton processes achieved ∼45 and ∼15 % of mineralization, respectively. During the electrochemical treatment, 100 % of the organic carbon remained even after the antibiotic was treated four times the time needed to degrade it. In contrast, in all processes, a natural matrix (mineral water) did not considerably inhibit pollutant elimination. However, the presence of glucose in the water significantly affected the degradation of CLX by means of TiO2 photocatalysis.