The functional repertoire of AmpR in the AmpC ß-lactamase high expression and decreasing ß-lactam and aminoglycosides resistance in ESBL Citrobacter freundii.

Citrobacter freundii is characterized by AmpC ß-lactamases that develop resistance to ß-lactam antibiotics. The production of extended-spectrum ß-lactamase (ESBL) is substantially high in Escherichia coli, C. freundii, Enterobacter cloacae, and Serratia marcescens, but infrequently explored in C. freundii. The present investigation characterized the ESBL C. freundii and delineated the genes involved in decrease in antibiotics resistance. We used the VITEK-2 system and Analytical Profile Index (API) kit to characterize and identify the Citrobacter isolates. The mRNA level of AmpC and AmpR was determined by RT-qPCR, and gel-shift assay was performed to evaluate protein-DNA binding. Here, a total of 26 Citrobacter strains were isolated from COVID-19 patients that showed varying degrees of antibiotic resistance. We examined and characterized the multidrug resistant C. freundii that showed ESBL production. The RT-qPCR analysis revealed that the AmpC mRNA expression is significantly high followed by a high level of AmpR. We sequenced the AmpC and AmpR genes that revealed the AmpR has four novel mutations in comparison to the reference genome namely; Thr64Ile, Arg86Ser, Asp135Val, and Ile183Leu while AmpC remained intact. The ΔAmpR mutant analysis revealed that the AmpR positively regulates oxidative stress response and decreases ß-lactam and aminoglycosides resistance. The AmpC and AmpR high expression was associated with resistance to tazobactam, ampicillin, gentamicin, nitrofurantoin, and cephalosporins whereas AmpR deletion reduced ß-lactam and aminoglycosides resistance. We conclude that AmpR is a positive regulator of AmpC that stimulates ß-lactamases which inactivate multiple antibiotics.

Ambulatory short-course high-dose oral amoxicillin for treatment of severe pneumonia in children: a randomised equivalency trial.

BACKGROUND: WHO case management guidelines for severe pneumonia involve referral to hospital for treatment with parenteral antibiotics. If equally as effective as parenteral treatment, home-based oral antibiotic treatment could reduce referral, admission, and treatment costs. Our aim was to determine whether home treatment with high-dose oral amoxicillin and inpatient treatment with parenteral ampicillin were equivalent for the treatment of severe pneumonia in children. METHODS: This randomised, open-label equivalency trial was done at seven study sites in Pakistan. 2037 children aged 3-59 months with severe pneumonia were randomly allocated to either initial hospitalisation and parenteral ampicillin (100 mg/kg per day in four doses) for 48 h, followed by 3 days of oral amoxicillin (80-90 mg/kg per day; n=1012) or to home-based treatment for 5 days with oral amoxicillin (80-90 mg/kg per day in two doses; n=1025). Follow-up assessments were done at 1, 3, 6, and 14 days after enrollment. The primary outcome was treatment failure (clinical deterioration) by day 6. Analyses were done per protocol and by intention to treat. This trial is registered, ISRCTN95821329. FINDINGS: In the per-protocol population, 36 individuals were excluded from the hospitalised group and 37 from the ambulatory group, mainly because of protocol violations or loss to follow-up. There were 87 (8.6%) treatment failures in the hospitalised group and 77 (7.5%) in the ambulatory group (risk difference 1.1%; 95% CI -1.3 to 3.5) by day 6. Five (0.2%) children died within 14 days of enrollment, one in the ambulatory group and four in the hospitalised group. In each case, treatment failure was declared before death and the antibiotic had been changed. None of the deaths were considered to be associated with treatment allocation; there were no serious adverse events reported in the trial. INTERPRETATION: Home treatment with high-dose oral amoxicillin is equivalent to currently recommended hospitalisation and parenteral ampicillin for treatment of severe pneumonia without underlying complications, suggesting that WHO recommendations for treatment of severe pneumonia need to be revised.

Deletion mutation in BSCL2 gene underlies congenital generalized lipodystrophy in a Pakistani family.

BACKGROUND: Congenital generalized lipodystrophy (CGL) also known as Berardinelli-Seip Congenital Lipodystrophy (BSCL) is a genetically heterogeneous disorder characterized by loss of adipose tissues, Acanthosis nigricans, diabetes mellitus, muscular hypertrophy, hepatomegaly and hypertriglyceridemia. There are four subclinical phenotypes of CGL (CGL1-4) and mutations in four genes AGPAT2, BSCL2, CAV1 and PTRF have been assigned to each type. METHODS: The study included clinical and molecular investigations of CGL disease in a consanguineous Pakistani family. For mutation screening all the coding exons including splice junctions of AGPAT2, BSCL2, CAV1 and PTRF genes were PCR amplified and sequenced directly using an automated DNA sequencer ABI3730. RESULTS: Sequence analysis revealed a single base pair deletion mutation (c.636delC; p.Tyr213ThrfsX20) in exon 5 of BSCL2 gene causing a frame shift and premature termination codon. CONCLUSION: Mutation identified here in BSCL2 gene causing congenital generalized lipodystrophy is the first report in Pakistani population. The patients exhibited characteristic features of generalized lipodystrophy, Acanthosis nigricans, diabetes mellitus and hypertrophic cardiomyopathy. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1913913076864247.