Resistance to amoxicillin-clavulanate, a widely used beta-lactam/beta-lactamase inhibitor combination antibiotic, is rising globally, yet susceptibility testing remains challenging. To test whether whole-genome sequencing (WGS) could provide a more reliable assessment of susceptibility than traditional methods, we predicted resistance from WGS for 976 E. coli bloodstream infection isolates from Oxfordshire, UK, comparing against phenotypes from the BD Phoenix (calibrated against EUCAST guidelines). 339/976 (35%) isolates were amoxicillin-clavulanate resistant. Predictions based solely on beta-lactamase presence/absence performed poorly (sensitivity 23% (78/339)) but improved when genetic features associated with penicillinase hyper-production (e.g. promoter mutations, copy number estimates) were considered (sensitivity 82% (277/339); p<0.0001). Most discrepancies occurred in isolates with peri-breakpoint MICs. We investigated two potential causes; the phenotypic reference and the binary resistant/susceptible classification. We performed reference standard, replicated phenotyping in a random stratified subsample of 261/976 (27%) isolates using agar dilution, following both EUCAST and CLSI guidelines, which use different clavulanate concentrations. As well as disagreeing with each other, neither agar dilution phenotype aligned perfectly with genetic features. A random-effects model investigating associations between genetic features and MICs showed that some genetic features had small, variable and additive effects, resulting in variable resistance classification. Using model fixed-effects to predict MICs for the non-agar dilution isolates, predicted MICs were in essential agreement (±1 doubling dilution) with observed (BD Phoenix) MICs for 691/715 (97%) isolates. This suggests amoxicillin-clavulanate resistance in E. coli is quantitative, rather than qualitative, explaining the poorly reproducible binary (resistant/susceptible) phenotypes and suboptimal concordance between different phenotypic methods and with WGS-based predictions.

Objectives: Carbapenemase-producing Enterobacterales and specifically KPC-producing Klebsiella pneumoniae (KPC-Kp) are rapidly spreading worldwide. The prognosis of ventilator-associated pneumonia (VAP) caused by KPC-producing Klebsiella pneumoniae (KPC-Kp) is not well known. Our study tries to assess whether ventilator-associated pneumonia caused by a KPC-Kp strain is associated with higher all-cause mortality than if caused by carbapenem-susceptible isolates.

Study design and methods: This is a retrospective cohort study of patients with VAP due to K. pneumoniae from a 35-bed polyvalent Intensive Care Unit in a university hospital (> 40,000 annual admissions) between January 2012 and December 2016. Adjusted multivariate analysis was used to study the association of KPC-Kp with 30-day all-cause mortality (Cox regression).

Results. We analyze 69 cases of K. pneumoniae VAP of which 39 were produced by a KPC-Kp strain with high-level resistance to meropenem (MIC > 16 mg/mL). All-cause mortality at 30 days was 41% in the KPC-Kp group (16/39) and 33.3% in the carbapenem-susceptible cases (10/30). KPC-Kp etiology was not associated with higher mortality when controlled for confounders (adjusted hazard ratio [lsqb]HR[rsqb] 1.25; 95% CI: 0.46–3.41). Adequate targeted therapy (HR 0.03; 95% CI: <0.01–0.23) was associated with all-cause mortality.

Conclussion. Assuming the limitations due to the available sample size, the prognosis of VAP caused by KPC-Kp is similar to VAPs caused by carbapenem-susceptible K. pneumoniae when appropriate treatment is used.

Highly conserved PenI-type class A β-lactamase in pathogenic members of Burkholderia can evolve to extended-spectrum β-lactamase (ESBL), which exhibits hydrolytic activity towards third-generation cephalosporins, while losing its activity towards the original penicillin substrates. We describe three single-amino-acid-substitution mutations in the ArgS arginine-tRNA synthetase that confer extra antibiotic tolerance protection to ESBL-producing Burkholderia thailandensis. This pathway can be exploited to evade antibiotic tolerance induction in developing therapeutic measures against Burkholderia species, targeting their essential aminoacyl-tRNA synthetases.

Periodontitis as a biofilm-associated inflammatory disease is highly prevalent worldwide. It severely affects oral health and yet closely links to systemic diseases like diabetes and cardiovascular disease. Porphyromonas gingivalis as a ‘keystone' periodontopathogen drives the shift of microbe-host symbiosis to dysbiosis, and critically contributes to the pathogenesis of periodontitis. Persisters are a tiny subset of biofilm-associated microbes highly tolerant to lethal treatment of antimicrobials, and notably metronidazole-tolerant P. gingivalis persisters have recently been identified by our group. This study further explored the interactive profiles of metronidazole-treated P. gingivalis persisters (M-PgPs) with human gingival epithelial cells (HGECs). P. gingivalis cells (ATCC 33277) at stationary phase were treated with lethal dosage of metronidazole (100 μg/ml, 6 hours) for generating M-PgPs. The interaction of M-PgPs with HGECs was assessed by microscopy, flow cytometry, cytokine profiling and qPCR. We demonstrated that the overall morphology and ultra-cellular structure of M-PgPs remained unchanged. Importantly, M-PgPs maintained the capabilities to adhere to and invade into HGECs. Moreover, M-PgPs significantly suppressed pro-inflammatory cytokine expression in HGECs at a comparable level with the untreated P. gingivalis cells, through the thermo-sensitive components. The present study reveals that P. gingivalis persisters induced by lethal treatment of antibiotics could maintain their capabilities to adhere to and invade into human gingival epithelial cells, and perturb the innate host responses. Novel strategies and approaches need to be developed for tackling P. gingivalis and favourably modulating the dysregulated immuno-inflammatory responses for oral/periodontal health and general wellbeing.

The Cpx stress response is widespread among Enterobacteriaceae. We have previously reported a mutation in cpxA in a multidrug resistant strain of Klebsiella aerogenes isolated from a patient treated with imipenem. This mutation yields to a single amino acid substitution (Y144N) located in the periplasmic sensor domain of CpxA. In this work, we sought to characterize this mutation in Escherichia coli by using genetic and biochemical approaches. Here, we show that cpxA^Y144N is an activated allele that confers resistance to β-lactams and aminoglycosides in a CpxR-dependent manner, by regulating the expression of the OmpF porin and the AcrD efflux pump, respectively. We also demonstrate the intimate interconnection between Cpx system and peptidoglycan integrity on the expression of an exogenous AmpC β-lactamase by using imipenem as a cell wall active antibiotic or inactivation of penicillin-binding proteins. Moreover, our data indicate that the Y144N substitution abrogates the interaction between CpxA and CpxP and increase phosphotransfer activity on CpxR. Because the addition of a strong AmpC inducer such as imipenem is known to causes abnormal accumulation of muropeptides (disaccharide-pentapeptide, N-acetylglucosamyl-1,6-anhydro-N-acetylmuramyl-l-alanyl-d-glutamy-meso-diaminopimelic-acid-d-alanyl-d-alanine) in the periplasmic space, we propose these molecules activate the Cpx system by displacing CpxP from the sensor domain of CpxA. Altogether, these data could explain why large perturbations to peptidoglycan caused by imipenem lead to mutational activation of the Cpx system and bacterial adaptation through multidrug resistance. These results also validate the Cpx system, in particular the interaction between CpxA and CpxP, as a promising therapeutic target.

QPX7728 is an ultra-broad-spectrum boronic acid beta-lactamase inhibitor that demonstrates inhibition of key serine and metallo beta-lactamases at a nano molar range in biochemical assays with purified enzymes. The broad-spectrum inhibitory activity of QPX7728 observed in biochemical experiments translates into enhancement of the potency of many beta-lactams against strains of target pathogens producing beta-lactamases. The impact of bacterial efflux and permeability on inhibitory potency were determined using isogenic panels of KPC-3 producing isogenic strains of K. pneumoniae and P. aeruginosa and OXA-23-producing strains of A. baumannii with various combinations of efflux and porin mutations. QPX7728 was minimally affected by multi-drug resistance efflux pumps in either Enterobacteriaceae, or in non-fermenters such as P. aeruginosa or A. baumannii. In P. aeruginosa, the potency of QPX7728 was further enhanced when the outer membrane is permeabilized. The potency of QPX7728 in P. aeruginosa is not affected by inactivation of the carbapenem porin OprD. While changes in OmpK36 (but not OmpK35) reduced the potency of QPX7728 (8-16-fold), QPX7728 (4 μg/ml) nevertheless completely reversed KPC-mediated meropenem resistance in strains with porin mutations, consistent with a lesser effect of these mutations on the potency of QPX7728 compared to other agents. The ultra-broad-spectrum beta-lactamase inhibition profile combined with enhancement of the activity of multiple beta-lactam antibiotics with varying sensitivity to the intrinsic resistance mechanisms of efflux and permeability indicate QPX7728 is a useful inhibitor for use with multiple beta-lactam antibiotics.

Polymyxins are increasingly used as the critical last-resort therapeutic options for multidrug-resistant gram-negative bacteria. Unfortunately, polymyxin resistance has increased gradually for the last few years. Although studies on mechanisms of polymyxin are expanding, system-wide analyses of the underlying mechanism for polymyxin resistance and stress response are still lacking. To understand how Klebsiella pneumoniae adapt to colistin (polymyxin E) pressure, we carried out proteomic analysis of Klebsiella pneumoniae strain cultured with different concentrations of colistin. Our results showed that the proteomic responses to colistin treatment in Klebsiella pneumoniae involving several pathways, including (i) gluconeogenesis and TCA cycle; (ii) arginine biosynthesis; (iii) porphyrin and chlorophyll metabolism; and (iv) enterobactin biosynthesis. Interestingly, decreased abundance of class A β-lactamases including TEM, SHV-11, SHV-4 were observed in cells treated with colistin. Moreover, we also present comprehensive proteome atlases of paired polymyxin-susceptible and -resistant Klebsiella pneumoniae strains. The polymyxin-resistant strain Ci, a mutant of Klebsiella pneumoniae ATCC BAA 2146, showed missense mutation in crrB. The crrB mutant Ci, which displayed lipid A modification with 4-amino-4-deoxy-L-arabinose (L-Ara4N) and palmitoylation, showed striking increases of CrrAB, PmrAB, PhoPQ, ArnBCADT and PagP. We hypothesize that crrB mutations induce elevated expression of the arnBCADTEF operon and pagP via PmrAB and PhoPQ. Moreover, multidrug efflux pump KexD, which was induced by crrB mutation, also contributed to colistin resistance. Overall, our results demonstrated proteomic responses to colistin treatment and the mechanism of CrrB-mediate colistin resistance, which may further offer valuable information to manage polymyxin resistance.

Antibiotic resistance is a global concern; however, data on antibiotic-resistant Ureaplasma spp. and Mycoplasma hominis are limited in comparison to similar data on other microbes. A total of 492 Ureaplasma spp. and 13 M. hominis strains obtained in Hangzhou, China, in 2018, were subjected to antimicrobial susceptibility testing for levofloxacin, moxifloxacin, erythromycin, clindamycin, and doxycycline using the broth microdilution method. The mechanisms underlying quinolone and macrolide resistance were determined. Meanwhile, a model of the topoisomerase IV complex bound to levofloxacin in wild-type Ureaplasma spp. was built to study the quinolone resistance mutations. For Ureaplasma spp., the levofloxacin, moxifloxacin and erythromycin resistance rates were 84.69%, 51.44% and 3.59% in U. parvum and 82.43%, 62.16% and 5.40% in U. urealyticum, respectively. Of the 13 M. hominis strains, 11 were resistant to both levofloxacin and moxifloxacin, and five strains showed clindamycin resistance. ParC S83L was the most prevalent mutation in levofloxacin-resistant Ureaplasma strains, followed by ParE R448K. The two mutations GyrA S153L and ParC S91I were commonly identified in quinolone-resistant M. hominis. A molecular dynamics-refined structure revealed that quinolone resistance-associated mutations inhibited the interaction and reduced affinity with gyrase or topoisomerase IV and quinolones. The novel mutations S21A in the L4 protein and G2654T and T2245C in 23S rRNA and ermB gene were identified in erythromycin-resistant Ureaplasma spp. Fluoroquinolone resistance in Ureaplasma spp. and Mycoplasma hominis remains high in China, the rational use of antibiotics needs to be further enhanced.

Klebsiella pneumoniae that produce extended spectrum beta lactamases (ESBLs) are a persistent public health threat. There are relatively few therapeutic options and there is undue reliance on carbapenems. Alternative therapeutic options are urgently required. A combination of cefepime and the novel beta lactamase inhibitor enmetazobactam is being developed for treatment of serious infections caused by ESBL-producing organisms. The pharmacokinetics-pharmacodynamics (PK-PD) of cefepime-enmetazobactam against ESBL-producing K. pneumoniae was studied in a neutropenic murine pneumonia model. Dose ranging studies were performed. Dose fractionation studies were performed to define the relevant PD index for the inhibitor. The partitioning of cefepime and enmetazobactam into the lung was determined by comparing area under the concentration time curve (AUC) in plasma and epithelial lining fluid. The magnitude of drug exposure for cefepime-enmetazobactam required for logarithmic killing in the lung was defined using 3 ESBL-producing strains. Cefepime 100 mg/kg q8h i.v. had minimal antimicrobial effect. When this background regimen of cefepime was combined with enmetazobactam half-maximal effect was induced with enmetazobactam 4.71 mg/kg q8h i.v. The dose fractionation study suggest both fT>threshold and fAUC:MIC are potentially relevant PD indices. The AUC_ELF:AUC_plasma for cefepime and enmetazobactam was 73.4% and 61.5%, respectively. A ≥2-log kill in the lung was achieved with a plasma and ELF cefepime fT>MIC of ≥20% and enmetazobactam fT>2 mg/L of ≥20% of the dosing interval. These data and analyses provide the underpinning evidence for the combined use of cefepime and enmetazobactam for nosocomial pneumonia.

The human diseases caused by the fungal pathogens Cryptococcus neoformans and C. gattii are associated with high indices of mortality, and toxic and/or cost-prohibitive therapeutic protocols. The need for affordable antifungals to combat cryptococcal disease is unquestionable. Previous studies suggested benzimidazoles as promising anti-cryptococcal agents combining low cost and high antifungal efficacy, but their therapeutic potential has not been demonstrated so far. In this study, we investigated the antifungal potential of fenbendazole, the most effective anti-cryptococcal benzimidazole. Fenbendazole was inhibitory against 17 different isolates of C. neoformans and C. gattii at a low concentration. The mechanism of anti-cryptococcal activity of fenbendazole involved microtubule disorganization, as previously described for human parasites. In combination with fenbendazole, the concentrations of the standard antifungal amphotericin B required to control cryptococcal growth were lower than those required when this antifungal was used alone. Fenbendazole was not toxic to mammalian cells. During macrophage infection, the anti-cryptococcal effects of fenbendazole included inhibition of intracellular proliferation rates and reduced phagocytic escape through vomocytosis. Fenbendazole deeply affected the cryptococcal capsule. In a mice model of cryptococcosis, the efficacy of fenbendazole to control animal mortality was similar to that observed for amphotericin B. These results indicate that fenbendazole is a promising candidate for the future development of an efficient and affordable therapeutic tool to combat cryptococcosis.

Clostridioides difficile, the leading cause of nosocomial infections, is an urgent health threat worldwide. The increased incidence and severity of disease, the high recurrence rates, and the dearth of effective anticlostridial drugs have created an urgent need for new therapeutic agents. In an effort to discover new drugs for treatment of Clostridioides difficile infections (CDIs), we investigated a panel of FDA-approved antiparasitic drugs against C. difficile and identified diiodohydroxyquinoline (DIHQ), an FDA-approved oral antiamoebic drug. DIHQ exhibited potent activity against 39 C. difficile isolates, inhibiting growth of 50% and 90% of these isolates at the concentrations of 0.5 μg/mL and 2 μg/mL, respectively. In a time-kill assay, DIHQ was superior to vancomycin and metronidazole, reducing a high bacterial inoculum by 3-log₁₀ within six hours. Furthermore, DIHQ reacted synergistically with vancomycin and metronidazole against C. difficile in vitro. Moreover, at subinhibitory concentrations, DIHQ was superior to vancomycin and metronidazole in inhibiting two key virulence factors of C. difficile, toxin production and spore formation. Additionally, DIHQ did not inhibit growth of key species that compose the host intestinal microbiota, such as Bacteroides, Bifidobacterium and Lactobacillus spp. Collectively, our results indicate that DIHQ is a promising anticlostridial drug that warrants further investigation as a new therapeutic for CDIs.

Chromosomal and plasmid-borne AmpC cephalosporinases are a major resistance mechanism to β-lactams in Enterobacteriaceae and Pseudomonas aeruginosa. The new β-lactamase inhibitor avibactam effectively inhibits class C enzymes and can fully restore ceftazidime susceptibility. The conserved amino acid residue Asn³⁴⁶ of AmpC cephalosporinases directly interacts with the avibactam sulfonate. Disruption of this interaction caused by the N³⁴⁶Y amino acid substitution in Citrobacter freundii AmpC was previously shown to confer resistance to the ceftazidime-avibactam combination (CAZ-AVI). The aim of this study was to phenotypically and biochemically characterize the consequences of the N³⁴⁶Y substitution in various AmpC backgrounds. Introduction of N³⁴⁶Y into Enterobacter cloacae AmpC (AmpC_cloacae), plasmid-mediated DHA-1, and P. aeruginosa PDC-5, led to 270-, 12,000-, and 79-fold decreases in the inhibitory efficacy (k₂/K_i) of avibactam, respectively. The kinetic parameters of AmpC_cloacaeand DHA-1 for ceftazidime hydrolysis were moderately affected by the substitution. Accordingly, AmpC_cloacaeand DHA-1 harboring N³⁴⁶Y conferred CAZ-AVI resistance (MIC of ceftazidime of 16 µg/ml in the presence of 4 µg/ml of avibactam). In contrast, production of PDC-5 N³⁴⁶Y was associated with a lower MIC (4 µg/ml) since this β-lactamase retained a higher inactivation efficacy by avibactam in comparison to AmpC_cloacaeN³⁴⁶Y. For FOX-3, the I³⁴⁶Y substitution did not reduce the inactivation efficacy of avibactam and the substitution was highly deleterious for β-lactam hydrolysis, including ceftazidime, preventing CAZ-AVI resistance. Since AmpC_cloacaeand DHA-1 display substantial sequence diversity, our results suggest that loss of hydrogen interaction between Asn³⁴⁶ and avibactam could be a common mechanism of acquisition of CAZ-AVI resistance.

Probiotics might provide an alternative approach for the control of oral candidiasis. However, studies on the antifungal activity of probiotics in the oral cavity are based on the consumption of yogurt or other dietary products, and there is a necessary to use appropriate biomaterials and specific strains to obtain probiotic formulations targeting local oral administration. In this study, we impregnated gellan gum, a natural biopolymer used as a food-additive, with a probiotic and investigated its antifungal activity against Candida albicans. Lactobacillus paracasei 28.4, a strain recently isolated from the oral cavity of a caries-free individual, was incorporated in several concentrations of gellan gum (0.6% to 1%). All tested concentrations could incorporate L. paracasei cells while maintaining bacterial viability. Probiotic/gellan formulations were stable for 7 days when stored at room temperature or 4°C. Long-term storage of bacteria-impregnated gellan gum was achieved when L. paracasei 28.4 was lyophilized. The probiotic/gellan formulations provided a release of L. paracasei cells over 24 hours that was sufficient to inhibit the growth of C. albicans with effects dependent on the cell concentrations incorporated into gellan gum. The probiotic/gellan formulations also had inhibitory activity against Candida spp. biofilms by reducing the number of Candida spp. cells (p < 0.0001), decreasing the total biomass (p = 0.0003), and impairing hyphae formation (p = 0.0002), compared to the control group which received no treatment. Interestingly, probiotic formulation of 1% w/v gellan gum provided an oral colonization of L. paracasei in mice with approximately 6 log of CFU/mL after 10 days. This formulation inhibited the C. albicans growth (p < 0.0001), prevented the development of candidiasis lesions (p = 0.0013), and suppressed inflammation (p = 0.0006) when compared to the mice not treated in the microscopic analysis of the tongue dorsum. These results indicate that gellan gum is a promising biomaterial and can be used as a carrier system to promote oral colonization for probiotics that prevent oral candidiasis.

Durlobactam (DUR, also known as ETX2514) is a novel β-lactamase inhibitor with broad activity against Ambler class A, C, and D β-lactamases. Addition of DUR to sulbactam (SUL) in vitro restores SUL activity against clinical isolates of Acinetobacter baumannii. The safety and pharmacokinetics (PK) of DUR alone and with SUL and/or imipenem/cilastatin (IMI/CIL) were evaluated in healthy subjects. This was a randomized, placebo-controlled study. In Part A, subjects including an elderly cohort (DUR 1 g) received single ascending doses of DUR 0.25-8 g. In Part B, multiple ascending dose of DUR 0.25-2 g were administered every 6 hours (q6h) for 29 doses. In Parts C and D, the drug-drug interaction (DDI) potential, including safety, of DUR (1 g) with SUL (1 g) and/or IMI/CIL (0.5/0.5 g) was investigated after single and multiple doses. Plasma and urine concentrations of DUR, SUL, and IMI/CIL were determined. Among 124 subjects, DUR was generally safe and well tolerated either alone or in combination with SUL and/or IMI/CIL. After single and multiple doses, DUR demonstrated linear dose proportional exposure across the studied dose ranges. Renal excretion was a predominant clearance mechanism. No drug:drug interaction potential was identified between DUR and SUL and/or IMI/CIL. SUL-DUR, 1 g (of each component) administered q6h with a 3 hour IV infusion, is under development for the treatment of serious infections due to A. baumannii.

Treatment of dogs naturally infected with Leishmania infantum using meglumine antimoniate (MA) encapsulated in conventional liposomes (LC) in association with allopurinol has been previously reported to promote marked reduction in the parasite burden in the main infection sites. Here, a new assay in naturally infected dogs was performed using a novel liposome formulation of MA consisting of a mixture of conventional and long-circulating (PEGylated) liposomes (LCP), with expected broader distribution among affected tissues of the mononuclear phagocyte system. Experimental groups of naturally infected dogs were as follows: LCP+Allop, receiving LCP intravenously as 2 cycles of 6 doses (6.5 mg Sb/kg/dose) at 4-day intervals, plus allopurinol at 30 mg/kg/12 h p.o. during 130 days; LC+Allop, receiving LC intravenously as 2 cycles of 6 doses (6.5 mg Sb/kg/dose), plus allopurinol during 130 days; Allop, treated with allopurinol only; non-treated control. Parasite loads were evaluated by quantitative PCR in liver, spleen and bone marrow and by immunohistochemistry in the ear skin, before, just after treatment and 4 months later. LCP+Allop and LC+Allop groups, but not the Allop group, showed significant suppression of the parasites in the liver, spleen and bone marrow 4 months after treatment, compared to the pre-treatment period or the control group. Only LCP+Allop group showed significantly lower parasite burden in the skin, in comparison to the control group. On the basis of clinical staging and parasitological evaluations, LCP formulation exhibited a more favorable therapeutic profile, when compared to LC one, being therefore promising for treatment of canine visceral leishmaniasis.

Attention has been paid to H5N6 highly pathogenic avian influenza virus (HPAIV) because of its heavy burden on the poultry industry and human mortality. Since an influenza A virus carrying N6 neuraminidase (NA) has never spread in humans, the potential for H5N6 HPAIV to cause disease in humans and the efficacy of antiviral drugs against the virus need to be urgently assessed. We used non-human primates to elucidate the pathogenesis of H5N6 HPAIV as well as to determine the efficacy of antiviral drugs against the virus. H5N6 HPAIV infection led to high fever in cynomolgus macaques. The lung injury caused by the virus was severe with diffuse alveolar damage and neutrophil infiltration. In addition, an increase in IFN-α showed an inverse correlation with virus titers during the infection process. Oseltamivir was effective for reducing H5N6 HPAIV propagation, and continuous treatment with peramivir reduced virus propagation and severity of symptoms in the early stage. This study also showed the pathologically severe lung injury states in the cynomolgus macaques infected with H5N6 HPAIV, even in those that received early antiviral drug treatments, indicating the need for close monitoring and the need for further studies on the virus pathogenicity and new antiviral therapies.

A total of 2,283 Salmonella spp. isolates were recovered from 18,334 samples including patients with diarrhea, food of animal origin and pets across 5 provinces of China. The highest prevalence of Salmonella spp. was detected in chicken meats (39.3%, 486/1,237). Fifteen serogroups and 66 serovars were identified, with Typhimurium and Enteritidis being the most dominant. Most (85.5%, 1,952/2,283) isolates exhibited resistant to ≥ 1 antimicrobial and 56.4% were multi-drug resistant (MDR). A total of 222 isolates harbored extended-spectrum β-lactamases (ESBLs), 200 of which were CTX-M-type that were mostly detected from chicken meat and turtle fecal. Overall, eight bla_CTX-M genes were identified, with bla_CTX-M-65, bla_CTX-M-123, bla_CTX-M-14, bla_CTX-M-79, and bla_CTX-M-130 being the most prevalent. Totally, 166 of the 222 ESBL-producing isolates had amino acid substitutions in GyrA (S83Y, S83F, D87G, D87N, and D87Y) and ParC (and S80I), whilst the PMQR-encoding genes oqxA/B, qepA, and qnrB/S were detected in almost all isolates. Of the fifteen sequence types (STs) identified in the 222 ESBLs, ST17, ST11, ST34, and ST26 ranked among the top 5 in the number of isolates. Our study revealed considerable serovars diversity, high prevalence of co-occurrence of MDR determinants, including CTX-M-type ESBLs, QRDRs mutations and PMQR genes. This is the first report of CTX-M-130 Salmonella spp. from patients with diarrhea and QRDRs mutations from turtle fecal samples. Our study emphasizes the importance of actions, both in the health care settings and in the veterinary medicine sector, to control the dissemination of MDR, especially the CTX-M Salmonella spp. isolates.

Seasonal and pandemic influenza causes 650,000 deaths annually in the world. The emergence of drug-resistance to specific anti-influenza drugs such as oseltamivir and baloxavir marboxil highlights the urgency of novel anti-influenza chemical entity discovery. In this study, we report a series of novel thiazolides derived from an FDA-approved drug nitazoxanide with antiviral activity against influenza and a broad range of viruses. The preferred candidates 4a and 4d showed significantly enhanced anti-influenza potentials with 10-fold improvement, compared with nitazoxanide, and were effective against a variety of influenza subtypes including oseltamivir-resistant strains. Notably, the combination using of compounds 4a/4d and oseltamivir carboxylate or zanamivir displayed synergistic antiviral effect against oseltamivir-resistant strain. Mode of action analysis demonstrated that compounds 4a/4d acted at the late phase of viral infection cycle through inhibiting viral RNA transcription and replication. Further experiments showed that treatment with compounds 4a/4d significantly inhibited influenza virus infection in human lung organoids, suggesting the druggability of the novel thiazolides. In-depth transcriptome analysis revealed a series of up-regulated cellular genes that may contribute to the antiviral activities of 4a/4d. Together, our study pointed the optimization direction of nitazoxanide as anti-influenza drug, and discovered two novel-structured candidates 4a/4d with relatively broad-spectrum antiviral potential.

Globally, mutations in the katG gene account for the majority of isoniazid-resistant strains of Mycobacterium tuberculosis. Buyankhishig et al analyzed a limited number of Mycobacterium tuberculosis strains in Mongolia and found that isoniazid resistance was mainly attributable to inhA mutations. The GenoType® MTBDRplus assay was performed for isolates collected in the First National Tuberculosis Prevalence Survey and the Third Anti-Tuberculosis Drug Resistance Survey to investigate genetic mutations associated with isoniazid resistance in Mycobacterium tuberculosis in Mongolia. Of the 409 isoniazid-resistant isolates detected by the GenoType® MTBDRplus assay, 127 (31.1%) were resistant to rifampicin, 294 (71.9%) had inhA mutations without katG mutations, 113 (27.6%) had katG mutations without inhA mutations, and two (0.5%) strains had mutations in both the inhA and katG genes. Of the 115 strains with any katG mutation, 114 (99.1%) had mutations in codon 315 (S315T). Of the 296 trains with any inhA mutation, 290 (98.0%) had a C–15T mutation. The proportion of isoniazid-resistant strains with katG mutations was 25.3% among new cases and 36.2% among retreatment cases (p=0.03), as well as 17.0% among rifampicin-susceptible strains and 52.8% among rifampicin-resistant strains (p<0.01). Rifampicin resistance was significantly associated with the katG mutation (adjusted odds ratio 5.36, 95% CI 3.3–8.67, p<0.001). Mutations in inhA predominated in isoniazid-resistant tuberculosis in Mongolia. However, the proportion of katG mutations in isolates from previously treated cases was higher than that among new cases, and that in cases with rifampicin resistance was higher than that in cases without rifampicin resistance.

Dihydroartemisinin-piperaquine has shown excellent efficacy and tolerability in malaria treatment. However, concerns have been raised of potentially harmful cardiotoxic effects associated with piperaquine. The population pharmacokinetics and cardiac effects of piperaquine were evaluated in 1,000 patients, mostly children enrolled in a multicentre trial from 10 sites in Africa. A linear relationship described the QTc-prolonging effect of piperaquine, estimating a 5.90ms mean QTc-prolongation per 100ng/mL increase in piperaquine concentration. The effect of piperaquine on absolute QTc-interval estimated a mean maximum QTc-interval of 456ms (EC₅₀=209ng/mL). Simulations from the pharmacokinetic-pharmacodynamic models predicted 1.98-2.46% risk of having QTc-prolongation > 60ms in all treatment settings. Although piperaquine administration resulted in QTc-prolongation, no cardiovascular adverse events were found in these patients. Thus, the use of dihydroartemisinin-piperaquine should not be limited by this concern.

Meropenem/vaborbactam resistance in Klebsiella pneumoniae is associated with loss of function mutations in the OmpK35 and OmpK36 porins. Here we identify two previously unknown loss of function mutations that confer cefuroxime resistance in K. pneumoniae. The proteins lost were NlpD and KvrA; the latter is a transcriptional repressor controlling capsule production. We demonstrate that KvrA loss reduces OmpK35 and OmpK36 porin production, which confers reduced susceptibility to meropenem/vaborbactam in a KPC-3 producing K. pneumoniae isolate.

Background: MRSA pose significant therapeutic challenges, related to their: frequency in clinical infections; innate virulence properties; and propensity for multi-antibiotic resistance. MRSA are among the most common causes of endovascular infections, including infective endocarditis (IE).

Objective: To employ transthoracic echocardiography (TTE) to evaluate the effect of exebacase, a novel direct lytic agent, in experimental aortic valve MRSA IE.

Study Design: TTE was utilized to evaluate the in vivo effect of exebacase on MRSA-infected vegetation progression when combined with daptomycin (vs daptomycin alone). Primary intravegetation outcomes were: maximum size; weights at sacrifice; and MRSA counts at infection baseline vs after 4 days of daptomycin treatment (alone or in addition to exebacase administered once on treatment Day 1).

Results: A single dose of exebacase in addition to daptomycin cleared significantly more intravegetation MRSA than daptomycin alone. This was associated with a statistical trend toward reduced maximum vegetation size in the exebacase + daptomycin vs the daptomycin-alone therapy groups (p = 0.07). Also, mean vegetation weights in the exebacase-treated group were significantly lower vs daptomycin-alone (p < 0.0001). Maximum vegetation size by TTE correlated with vegetation weight (p = 0.005). In addition, intravegetation MRSA counts in the combination group were significantly lower vs untreated controls (p<0.0001) and the daptomycin-alone group (p<0.0001).

Conclusion: This study suggests that exebacase has a salutary impact on MRSA-infected vegetation progression when combined with daptomycin, especially in terms of vegetation MRSA burden, size and weight. Moreover, TTE appears to be an efficient non-invasive tool to assess therapeutic efficacies in experimental MRSA IE.

Many transferable quinolone-resistance mechanisms have been already identified in Gram-negative bacteria. The plasmid-encoded 65 amino-acid long ciprofloxacin-modifying enzyme, namely CrpP, was recently identified in Pseudomonas aeruginosa. We analyzed a collection of 100 clonally-unrelated and multidrug-resistant P. aeruginosa clinical isolates among which 46 (46%) were found positive for crpP-like genes, encoding five CrpP variants conferring variable levels of reduced susceptibility to fluoroquinolones. Those crpP-like genes were chromosomally located, as part of PAGI-like pathogenicity genomic islands.

Chagas disease, caused by the protozoan Trypanosoma cruzi, is one of the main causes of death due to cardiomyopathy and heart failure in Latin American countries. The treatment of Chagas disease is directed at eliminating the parasite, decreasing the probability of cardiomyopathy, and disrupting the disease transmission cycle. Benznidazole (BZ) and nifurtimox (NFX) are recognized as effective drugs for the treatment of Chagas disease by the World Health Organization, but both have high toxicity and limited efficacy, especially in the chronic disease phase. At low doses, aspirin (ASA) has been reported to protect against T. cruzi infection. We evaluated the effectiveness of BZ in combination with ASA at low doses during the acute disease phase and evaluated cardiovascular aspects and cardiac lesions in the chronic phase. ASA treatment prevented the cardiovascular dysfunction (hypertension and tachycardia) and typical cardiac lesions. Moreover, BZ+ASA-treated mice had a smaller cardiac fibrotic area than that in BZ-treated mice. These results were associated with an increase in the number of eosinophils and reticulocytes and level of nitric oxide in the plasma and cardiac tissue of ASA-treated mice relative to respective controls. These effects of ASA and BZ+ASA in chronically infected mice were inhibited by pretreatment with the LXA₄ receptor antagonist, Boc-2, indicating that the protective effects of ASA are mediated by ASA-triggered lipoxin. These results emphasize the importance of exploring new drug combinations for treatments of acute phase of Chagas disease that are beneficial for chronic patients.

Florfenicol belongs to a class of phenicol antimicrobials widely used as feed additives and for the treatment of respiratory infections. In recent years, increasing resistance to florfenicol has been reported in Campylobacter spp., the leading foodborne enteric pathogen causing diarrheal diseases worldwide. Here, we reported the identification of fexA, a novel mobile florfenicol resistance gene in Campylobacter. Of the 100 Campylobacter jejuni strains isolated from poultry in Zhejiang, China, nine of them were shown to be fexA positive, and their whole genome sequences were further determined by integration of Illumina short-read and MinION long-read sequencing. The fexA gene was found in the plasmid of one strain and chromosomes of eight strains, and its location was verified by S1 nuclease pulsed-field gel electrophoresis (S1-PFGE) and Southern blotting. Based on comparative analysis, the fexA gene was located within a region with the tet(L)-fexA-catA-tet(O) gene arrangement, demonstrated to be successfully transferrable among C. jejuni strains. Functional cloning indicated that acquisition of the single fexA gene significantly increased resistance to florfenicol, whereas its inactivation resulted in increased susceptibility to florfenicol in Campylobacter. Taken together, these results indicated that the emerging fexA resistance is horizontally transferable, which might greatly facilitate the adaptation of Campylobacter in food producing environments where florfenicols are frequently used.

Methicillin-resistant Staphylococcus aureus (MRSA) has grown to become a major burden on healthcare systems. The cumulation of limited therapeutic options and worsened patient outcomes with persistent MRSA bacteremia has driven research in optimizing its initial management. The guidelines published by the Infectious Disease of America currently recommend combination therapy for refractory MRSA bacteremia, but the utility of combining antibiotics from the start of therapy is under investigation. The alternative strategy of early use of a β-lactam antibiotics in combination with vancomycin upon initial MRSA bacteremia detection has shown promise. While this concept has gained international attention, providers should give this strategy serious consideration prior to implementation. The objective of this review is to examine retrospective and prospective evidence for early combination with vancomycin and β-lactam antibiotics, as well as explore potential consequences of combination therapy.

Treatment of exacerbations of chronic Pseudomonas aeruginosa infections in patients with cystic fibrosis (CF) is highly challenging due to hypermutability, biofilm formation and an increased risk of resistance emergence. We evaluated the impact of ciprofloxacin and meropenem as monotherapy and in combination in the dynamic in vitro CDC biofilm reactor (CBR). Two hypermutable P. aeruginosa strains, PAOmutS (MIC_{ciprofloxacin} 0.25 mg/L, MIC_meropenem 2 mg/L) and CW44 (MIC_{ciprofloxacin} 0.5 mg/L, MIC_meropenem 4 mg/L), were investigated for 120h. Concentration-time profiles achievable in epithelial lining fluid (ELF) following FDA-approved doses were simulated in the CBR. Treatments were ciprofloxacin 0.4g every 8h as 1h-infusions (80% ELF penetration), meropenem 6 g/day as continuous infusion (CI; 30% and 60% ELF penetration) and their combinations. Counts of total and less-susceptible planktonic and biofilm bacteria and MICs were determined. Antibiotic concentrations were quantified by UHPLC-PDA. For both strains, all monotherapies failed with substantial regrowth and resistance of planktonic (≥8log₁₀ CFU/mL) and biofilm (>8log₁₀ CFU/cm²) bacteria at 120h (MIC_{ciprofloxacin} up to 8 mg/L, MIC_meropenem up to 64 mg/L). Both combination treatments demonstrated synergistic bacterial killing of planktonic and biofilm bacteria of both strains from ~48h onwards and suppressed regrowth to ≤4log₁₀ CFU/mL and ≤6log₁₀ CFU/cm² at 120h. Overall, both combination treatments suppressed amplification of resistance of planktonic bacteria for both strains, and biofilm bacteria for CW44. The combination with meropenem at 60% ELF penetration also suppressed amplification of resistance of biofilm bacteria for PAOmutS. Thus, combination treatment demonstrated synergistic bacterial killing and resistance suppression against difficult-to-treat hypermutable P. aeruginosa strains.

Peroxidases are a group of heterogeneous family of enzyme that plays diverse biological functions. Ascorbate peroxidase is a redox enzyme that is reduced by trypanothione, which plays a central role in the redox defence system of Leishmania. In view of developing new and novel therapeutics, we have performed in silico studies in order to search for ligand library and identification of new drug candidates and its physiological role against promastigotes and intracellular amastigotes of Leishmania donovani. Our results demonstrated that the selected inhibitor ZINC96021026 has significant anti-leishmanial effect and effectively killed both free and intracellular forms of the parasite. ZINC96021026 was found to be identical to ML-240, a selective inhibitor of Valosin-containing protein (VCP) or p97, a member of AAA-ATPase protein family which was derived from the scaffold of DBeQ, targeting the D2-ATPase domain of the enzyme. ZINC96021026 (ML-240) thus have broad range of cellular functions, thought to be derived from its ability to unfold proteins or disassemble protein complexes besides inhibiting the ascorbate peroxidase activity. ML-240 may inhibits the parasite's ascorbate peroxidase leading to extensive apoptosis and inducing generation of reactive oxygen species. Taken together, our results demonstrated that ML-240 could be an attractive therapeutic option for treatment against leishmaniasis.

The off-label use of third generation cephalosporin (3GC) during in ovo vaccination or vaccination of newly hatched chicks, was a common practice worldwide. CMY-2-producing Escherichia coli have been disseminated among broiler production. The objectives of this study were to determine the epidemiological linkage of bla_CMY-2-positive plasmids among broilers both within and outside Japan because grandparent stock and parent stock were imported in Japan. We examined the whole genome sequences of 132 3GC-resistant E. coli isolates collected from healthy broilers during 2002-2014. The predominant 3GC-resistance gene was bla_CMY-2, which was detected in the plasmids of 87 (65.9%) isolates. The main plasmid replicon types were IncI1-I (n=21; 24.1%), IncI (n=12; 13.8%), IncB/O/K/Z (n=28; 32.2%), and IncC (n=22; 25.3%). Those plasmids were subjected to gene clustering and network analyses and plasmid multi-locus sequence typing (pMLST). The chromosomal DNA of isolates was subjected to MLST and single nucleotide variant (SNV)-based phylogenetic analysis.

MLST and SNV-based phylogenetic analysis revealed high diversity of E. coli isolates. ST429 harboring bla_CMY-2-positive IncB/O/K/Z was closely related to isolates from broiler in Germany harboring bla_CMY-2-positive IncB/O/K/Z. pST55-IncI and pST12-IncI1-I and pST3-IncC were prevalent in western Japan. pST12-IncI1-I and pST3-IncC were closely related to those detected in E. coli isolates from chicken in American continent, whereas 26 IncB/O/K/Z were related to those in Europe. These data will be useful to reveal the whole picture of transmission of CMY-2-producing bacteria in and out of Japan.

Background

Optimal concentrations of unbound antimicrobials are essential for maximum microbiological effect. Although hypoalbuminemia and albumin fluid resuscitation are common in critical care, the effects of different albumin concentrations on the unbound concentrations of highly protein-bound antimicrobials are not known. The aim of this study was to compare effects of different albumin states on total and unbound concentrations of ertapenem and ceftriaxone using an ovine model.

Methods

Design

Prospective, three phase intervention observational study.

Subjects

Healthy Merino sheep.

Interventions

Eight sheep were subject to three experimental phases; normoalbuminemia, hypoalbuminemia using plasmapheresis and albumin replacement using a 25% albumin solution. In each phase, ceftriaxone 40 mg/kg and ertapenem 15 mg/kg were given intravenously. Blood samples were collected at pre-defined intervals and analyzed using an ultra-high-performance liquid chromatography tandem mass spectrometry method. Pharmacokinetic parameters such as area under the curve (AUC_0-24), plasma clearance (CL) and apparent volume of distribution in the terminal phase (V_d) were estimated and compared between the phases.

Results

The protein and albumin concentrations were significantly different between phases. Hypoalbuminemia resulted in a significantly lower AUC_0-24 and higher CL of total and unbound concentrations of ceftriaxone compared to the other phases. Whereas albumin replacement led to higher AUC_0-24 and lower CL compared to other phases for both drugs. The V_d for total drug concentrations for both drugs were significantly lower with albumin replacement.

Conclusions

For highly protein-bound drugs such as ceftriaxone and ertapenem, both hypoalbuminemia and albumin replacement may affect unbound drug exposure.

Global markets rallied on the back of optimism that most governments were gearing up to at least partially reopen their economies following lockdowns to prevent the spread of the coronavirus.

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BACKGROUND:

The ability of the decades-old Boston and Philadelphia criteria to accurately identify infants at low risk for serious bacterial infections has not been recently reevaluated.

BACKGROUND AND OBJECTIVES:

Antibiotic therapy is often prescribed for suspected community-acquired pneumonia (CAP) in children despite a lack of knowledge of causative pathogen. Our objective in this study was to investigate the association between antibiotic prescription and treatment failure in children with suspected CAP who are discharged from the hospital emergency department (ED).

BACKGROUND:

Orphan drug development is crucial for children, who are disproportionately affected by rare diseases. Data are lacking on the number, nature, and benefit of recently approved pediatric orphan indications.

BACKGROUND:

For children who cannot be discharged from the emergency department, definitive care has become less frequent at most hospitals. It is uncertain whether this is true for common conditions that do not require specialty care. We sought to determine how the likelihood of definitive care has changed for 3 common pediatric conditions: asthma, croup, and gastroenteritis.

BACKGROUND:

Approximately 25% of children with concussion have persistent postconcussive symptoms (PPCS) with resultant significant impacts on quality of life. Melatonin has significant neuroprotective properties, and promising preclinical data suggest its potential to improve outcomes after traumatic brain injury. We hypothesized that treatment with melatonin would result in a greater decrease in PPCS symptoms when compared with a placebo.

BACKGROUND:

Mexican-origin women breastfeed at similar rates as white women in the United States, yet they usually breastfeed for less time. In our study, we seek to identify differences in Mexican-origin women’s breastfeeding intentions, initiation, continuation, and supplementation across nativity and country-of-education groups.

BACKGROUND:

Congenital diaphragmatic hernia (CDH) is a rare congenital anomaly with a mortality of ~27%. The Congenital Diaphragmatic Hernia Study Group (CDHSG) developed a simple postnatal clinical prediction rule to predict mortality in newborns with CDH. Our aim for this study is to externally validate the CDHSG rule in the European population and to improve its prediction of mortality by adding prenatal variables.

BACKGROUND:

Elevated non–high-density lipoprotein cholesterol (HDL-C) levels are used to identify children at increased cardiovascular risk, but the use of non–HDL-C in childhood to predict atherosclerosis is unclear. We examined whether the National Heart, Lung, and Blood Institute classification of youth non–HDL-C status predicts high common carotid artery intima-media thickness in adulthood.

OBJECTIVES:

Infantile hemangiomas (IHs) are common; some cases require timely referral and treatment to prevent complications. We developed and validated a reliable instrument for timely and adequate referral of patients with IH to experts by nonexpert primary physicians.

BACKGROUND:

Current guidelines from the American Academy of Pediatrics recommend screening children for developmental problems by using a standardized screening tool and referring at-risk patients to early intervention (EI) or subspecialists. Adoption of guidelines has been gradual, with research showing many children still not being screened and referred.

BACKGROUND:

Pediatricians are less frequently sued than other physicians. When suits are successful, however, the average payout is higher. Little is known about changes in the risk of litigation over time. We sought to characterize malpractice lawsuit trends for pediatricians over time.

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