Assessment of three antibiotic combination regimens against Gram-negative bacteria causing neonatal sepsis in low- and middle-income countries.

Gram-negative bacteria (GNB) are a major cause of neonatal sepsis in low- and middle-income countries (LMICs). Although the World Health Organization (WHO) reports that over 80% of these sepsis deaths could be prevented through improved treatment, the efficacy of the currently recommended first- and second-line treatment regimens for this condition is increasingly affected by high rates of drug resistance. Here we assess three well known antibiotics, fosfomycin, flomoxef and amikacin, in combination as potential antibiotic treatment regimens by investigating the drug resistance and genetic profiles of commonly isolated GNB causing neonatal sepsis in LMICs. The five most prevalent bacterial isolates in the NeoOBS study (NCT03721302) are Klebsiella pneumoniae, Acinetobacter baumannii, E. coli, Serratia marcescens and Enterobacter cloacae complex. Among these isolates, high levels of ESBL and carbapenemase encoding genes are detected along with resistance to ampicillin, gentamicin and cefotaxime, the current WHO recommended empiric regimens. The three new combinations show excellent in vitro activity against ESBL-producing K. pneumoniae and E. coli isolates. Our data should further inform and support the clinical evaluation of these three antibiotic combinations for the treatment of neonatal sepsis in areas with high rates of multidrug-resistant Gram-negative bacteria.

Klebsiella Species and Enterobacter cloacae Isolates Harboring blaOXA-181 and blaOXA-48: Resistome, Fitness Cost, and Plasmid Stability.

IncX3 and IncL plasmids have been named as catalysts advancing dissemination of blaOXA-181 and blaOXA-48 genes. However, their impact on the performance of host cells is vastly understudied. Genetic characteristics of blaOXA-48- and blaOXA-181-containing Klebsiella pneumoniae (EFN299), Klebsiella quasipneumoniae (EFN262), and Enterobacter cloacae (EFN743) isolated from clinical samples in a Ghanaian hospital were investigated by whole-genome sequencing. Transfer of plasmids by conjugation and electroporation, plasmid stability, fitness cost, and genetic context of blaOXA-48, blaOXA-181, and blaDHA-1 were assessed. blaOXA-181 was carried on two IncX3 plasmids, an intact 51.5-kb IncX3 plasmid (p262-OXA-181) and a 45.3-kb IncX3 plasmid (p743-OXA-181) without replication protein sequence. The fluoroquinolone-resistant gene qnrS1 region was also excised, and unlike in p262-OXA-181, the blaOXA-181 drug-resistant region was not found on a composite transposon. blaOXA-48 was carried on a 74.6-kb conjugative IncL plasmid with unknown ~10.9-kb sequence insertion. This IncL plasmid proved to be highly transferable, with a conjugation efficiency of 1.8 × 10-2. blaDHA-1 was present on an untypeable 22.2 kb genetic structure. Plasmid stability test revealed plasmid loss rate between 4.3% and 12.4%. The results also demonstrated that carriage of IncX3-blaOXA-181 or IncL-blaOXA-48 plasmids was not associated with any fitness defect, but rather an enhanced competitive ability of host cells. This study underscores the significant contribution of IncX3 and IncL plasmids in the dissemination of resistance genes and their efficient transfer calls for regular monitoring to control the expansion of resistant strains. IMPORTANCE The growing rate of antibiotic resistance is an important global health threat. This threat is exacerbated by the lack of safe and potent alternatives to carbapenems in addition to the slow developmental process of newer and effective antibiotics. Infections by carbapenem-resistant Gram-negative bacteria are becoming almost untreatable, leading to poor clinical outcomes and high mortality rates. OXA-48-like carbapenemases are one of the most widespread carbapenemases accounting for resistance among Enterobacteriaecae. We characterized OXA-48- and OXA-181-producing Enterobacteriaecae to gain insights into the genetic basis and mechanism of resistance to carbapenems. Findings from the study showed that the genes encoding these enzymes were carried on highly transmissible plasmids, one of which had sequences absent in other similar plasmids. This implies that mobile genetic elements are important players in the dissemination of resistance genes. Further characterization of this plasmid is warranted to determine the role of this sequence in the spread of resistance genes.

Removal of diclofenac by a local bacterial consortium: UHPLC-ESI-MS/MS analysis of metabolites and ecotoxicity assessment.

Diclofenac (DCF) belongs to the class of nonsteroidal anti-inflammatory drugs, which is one of the most consumed by population and detected in raw sewage. Several studies have reported variable removal rates by biodegradation of diclofenac in wastewater treatment plants (WWTPs). This study deals with the evaluation of the biodegradation of DCF by a bacterial consortium (obtained from pure cultures of Enterobacter hormaechei D15 and Enterobacter cloacea D16), which were isolated from household compost and Algerian WWTP, respectively, as sole carbon source and by co-metabolism, using glucose as carbon source. A 98% removal rate of DCF was observed when it is used as the sole carbon source, whilst only 44% of DCF was removed in co-metabolic conditions. Two metabolites were identified using ultra-high-performance liquid chromatography coupled to electrospray injection tandem mass spectrometry analysis (UHPLC-ESI-MS/MS); one of them was identified as 4'-hydroxy-DCF, and the second metabolite was suspected to be a nitro derivative of DCF, according to comparison with the literature. Biodegradation of DCF by this bacterial consortium generates relatively safe final by-products.

A simple and economic preservation method for genomic bacterial DNA from clinically significant pathogens.

Bacterial culture was allowed to dry to completeness on Columbia agar base with defibrinated horse blood. Following 6 months storage at room temperature, microbial DNA was extracted and successfully amplified by PCR. This storage technique has the advantage over other methods of not requiring (i) a DNA extraction protocol prior to storage and (ii) refrigeration and/or freezing. This technique maybe useful in the transportation of bacterial genomic DNA in nonviable cells as well as reliable method for the storage of DNA in underdeveloped countries.

Assessment of bacterial community structure in soil by polymerase chain reaction and denaturing gradient gel electrophoresis.

Bacterial community structure was studied in a Flevo silt loam (FSL) soil microplot, as well as in 15 other soils, by using DNA extraction followed by molecular fingerprinting. Total community DNA was extracted and purified by a direct method, which yielded amplifiable DNA of high molecular weight for all soils. A variable region of the 16S rRNA gene was then amplified by PCR with bacterial primers, resulting in a mixture of amplicons separable via denaturing gradient gel electrophoresis (DGGE). The DGGE profiles of FSL soil were indicative of dominant soil bacterial types, as evidenced by assessing the amplification of Enterobacter cloacae and Arthrobacter sp. targets in a soil DNA background. These targets produced barely detectable bands when present in soil DNA at roughly 5 x 10(6) genome equivalents per g dry soil, and strong bands at 27-fold higher levels. The PCR-DGGE analysis of the FSL soil was highly reproducible. Furthermore, different single versus composite topsoil samples yielded similar DGGE profiles with respect to major bands. In addition, samples taken along vertical soil cores (0-45 cm depth) revealed relative stability of the DGGE profiles. The profiles produced with DNA obtained from different aggregate size fractions of this soil were also similar with respect to the main bands. Moreover, FSL topsoil samples taken over a 1-year period (fallow soil) yielded stable profiles. These data suggested that the soil bacterial communities thus determined were dominated by a limited number of stable and ubiquitous types. The 16 soils, representing varying types and geographical locations, were assessed for differences in their bacterial DGGE profiles. There were striking differences between the profiles obtained for these soils. Evidence was found for the hypothesis that similar soil types tend to contain similar structures of the dominating bacterial types as revealed by the DGGE profiles.