Hospital wastewater can serve as a reservoir of ARB and antibiotic resistance genes (ARGs) due to massive quantities of antibiotics used in hospitals for prevention and treatment purposes. Apart from the high bacterial loads commonly reported in hospital effluents, they also contain sub-therapeutic antibiotic concentrations. The hospital setting and its resultant waste can be regarded as significant hot spots for the presence and potential distribution of antibiotic-resistant bacteria (ARB). These results indicate that hospital wastewater is laden with potentially pathogenic MDR Enterobacteriaceae with various antibiotic resistance genes that can be spread to humans throughout the food chain, provided the wastewaters are not properly treated before eventual discharge into the environment. The frequencies of the non-β-lactam genes detected were catII, tetA, sulI and sulII, tetB, aadA, tetC, and tetD. The pAmpC resistance determinants detected were bla CIT, bla FOX, bla EBC, and bla DHA. Of the phenotypically resistant isolates screened for each class of antibiotics, the ESBLs detected were bla CTX-M group (including groups 1, 2, and 9), bla TEM, bla OXA-1-like, bla SHV, bla PER, bla VEB, bla OXA-48-like and bla VIM, and bla IMP. Molecular characterization via the Polymerase Chain Reaction uncovered various ESBLs, pAmpCs, and other non-β-lactam encoding genes. The generated multiple antibiotic resistance indices ranged between 0.5 to 1.0, with the highest value seen in one K. Notably, all the bacterial species displayed resistance against cefotaxime, ampicillin, nalidixic acid, and trimethoprim-sulfamethoxazole. All the bacterial species exhibited resistance phenotypically against at least four antibiotic classes and were classified as MDR. We established the antibiotic susceptibility profiles of these identified bacterial isolates against a panel of 18 selected antibiotics belonging to 11 classes were established following established guidelines by the Clinical and Laboratory Standard Institute. The other identified isolates included Citrobacter freundii and Escherichia coli (10%), Enterobacter asburiae (6%), Enterobacter amnigenus, Enterobacter hormaechei, and Enterobacter kobei (3%). Thirty-six percent of these belonged to Klebsiella oxytoca, while 29% were Klebsiella pneumoniae.

From this, 31 were identified as the targeted Enterobacteriaceae members. Using the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF), we identified a total of 44 presumptive Enterobacteriaceae isolates.

We collected samples from two hospitals in Amathole and Chris Hani District Municipalities in the Eastern Cape Province, South Africa, within October and November 2017. Therefore, we aimed to evaluate the occurrence of ESBL/pAmpC genetic determinants, and some essential non-β-lactam genetic determinants in the MDR phenotypic antimicrobial resistance in Enterobacteriaceae isolates recovered from hospital wastewater. We also strongly urge researchers to report as much information about their assays as possible in their publications.Globally, there is an increasing occurrence of multidrug-resistant (MDR) Enterobacteriaceae with extended-spectrum β-lactamases (ESBLs) and/or plasmid-encoded AmpC (pAmpC) β-lactamases in clinical and environmental settings of significant concern. We present an overview of the main steps in the primer design workflow, with data that illustrate some of the unexpected variability that often occurs when theory is translated into practice. Despite the framework provided by the MIQE guidelines and the accessibility of wide-ranging support from peer-reviewed publications, books and online sources as well as commercial companies, the design of many published assays continues to be less than optimal: primers often lack intended specificity, can form dimers, compete with template secondary structures at the primer binding sites or hybridise only within a narrow temperature range. Consequently, poor design combined with failure to optimise reaction conditions is likely to result in reduced technical precision and false positive or negative detection of amplification targets. Primers are arguably the single most critical components of any PCR assay, as their properties control the exquisite specificity and sensitivity that make this method uniquely powerful.

Enzymex primer verification