Two hundred (200) clinical, non-duplicate Gram-negative bacterial pathogens were randomly selected from various human samples routinely processed by the hospital diagnostic microbiology laboratory. The emergence and spread of antibiotic resistance is a burden on both communities and healthcare systems in both developed and developing countries, with serious implications for the prevention and treatment of infections (Carlet & Pittet, 2013; Spellberg & Gilbert, 2014; Ventola, 2015). Clinical gram-negative 'ESKAPE' bacteria (K. pneumoniae, A. baumannii, P. aeruginosa and Enterobacter spp) are associated with high morbidity and mortality and are mainly involved in nosocomial infections (Flynn et al., 2015; Paramythiotou & Routsi, 2016 ;.
These pathogens are reported to be increasingly resistant to all clinically available antibiotic classes, including β-lactams (especially carbapenems and third- and fourth-generation cephalosporins), fluoroquinolones, aminoglycosides, and to some extent polymyxin B (colistin), often used as a last resort resort to antibiotics (Navidinia, 2016; Lim et al., 2011). A number of studies in Ghana on antibiotic resistance among Gram-negative bacteria, including some ESKAPE pathogens, have been conducted in hospitals, but many of the studies were centered at the Korle-Bu Teaching Hospital in the Greater Accra region of the country (Hackman et al. , 2014; Obeng-Nkrumah et al., 2013).
LITERATURE REVIEW
Advent of Antibiotics and Resistance
Mechanisms of Action of antibiotics
These agents bind to the amino acid acyl-D-alanyl-D-alanine thereby preventing the addition of new units to peptidoglycan, thereby causing murein hydrolases or transpeptidases to lyse the cell (Lupoli et al., 2011; Münch et al., 2015 ). These antibiotics bind to peptidoglycan units and interfere with transglycosylase and transpeptidase activity in the growing bacterial cell leading to inhibition or cell death (Kahne et al., 2005). From the mechanisms of action, these interfere with the normal cellular metabolism of bacteria, leading to growth inhibition or death of the organism (Giedraitienė et al., 2011; Huang, Zhu, & Melançon, 2015).
For example, DNA gyrase (DNA topoisomerase type II) and RNA polymerase are inhibited by quinolones and rifampicin respectively, preventing DNA overcoiling which interferes with the cellular processes of DNA synthesis, compromising thus the reproduction and survival of bacterial cells (Giedraitien, al. 2011; Zhou et al., 2015). Polymyxin B or colistin, a cyclic peptide antibiotic disrupts the bacterial cell membrane by interacting with phospholipids thereby increasing membrane permeability causing the cell to absorb excess water to promote bacterial death (Velkov, Roberts, Nation, Thompson, & Li, 201). .
Causes of bacterial resistance to antibiotics
The high prevalence of antibiotic resistance in Africa is largely due to the indiscriminate and inappropriate use of antibiotics in healthcare settings and communities (Donkor et al., 2012; Kiguba et al., 2016). Particularly in the outpatient setting, patient expectations regarding antibiotics are reported to be a major driving factor for overprescribing by physicians (Huttner et al., 2013). In many parts of the world, self-medication with antibiotics typically occurs outside the healthcare system, with more than 50% purchased and used without a prescription (Ocan et al., 2015).
This has resulted in many cases of misuse of antibiotics, such as incomplete course of treatment and inadequate dosing (Ocan et al., 2015), which are the main driving factors for the development of resistance. A study in the United Kingdom found significant levels of genes for resistance to clinically important antibiotics, including tetracyclines, sulfonamides, and trimethoprim, isolated from water bodies fed with wastewater from livestock farms where antibiotics had been used (Rowe et al., 2016).
Resistance Mechanisms of Gram-negative ESKAPE pathogens
Enzymes catalyze antibiotic inactivation by cleaving the amide bond of the β-lactam ring (acetylation reaction), rendering it ineffective in the bacterial cell (King, Sobhanifar, & Strynadka, 2017; Thenmozhi et al., 2014). Carbapenemases comprise chromosomal (IMI-1, NmcA, SFC-1, SME-1) and plasmid-encoded (KPC-2, GES, IMI-2, derivatives) carbapenemase genes and can hydrolyze all β-lactams, including monobactams (aztreonam). . however, they are inhibited or partially inhibited by β-lactamase inhibitors such as sulbactam, tazobactam, or clavulanic acid, but not by ethylene diamine tetraacetic acid [EDTA] ( Giedraitienė et al., 2011 ; Rice, 2010 ). MBLs usually consist of imipenemase metallo-β-lactamases (IMP), German imipenemases (GIM), Seoul imipenemases (SIM), Verona integron-encoded metallo-β-lactamases (VIM), and New Delhi metallo-β-lactamase-1. (NDM-1) enzymes with coding genes located on a plasmid or transposons and therefore easily spread between bacteria (Dahiya et al., 2015; Rice, 2010).
Group C consists of penicillinases and cephalosporinases, such as β-lactamase AmpC, which show higher hydrolytic activity against early cephalosporins than benzylpenicillin (Jeon et al., 2015). Carbapenems are mostly stable to AmpC β-lactamases (Thenmozhi et al., 2014), but in some bacteria, including A.
Clinical importance of Gram-negative ESKAPE pathogens
The most significant enzymes include ESBLs and carbapenemases that hydrolyze penicillins, cephalosporins, and carbapenems (King et al., 2017; Thenmozhi et al., 2014). The ESBLs are commonly identified as transferable β-lactamases in the pathogen and are inhibited by clavulanic acid, tazobactam or sulbactam (Shaikh et al., 2015; Swain & Padhy, 2016). The enzymatic inactivation of DNA gyrase or topoisomerase (IV) encoded by gyrA and parC genes in the bacterium combined with the expression of efflux pumps confers resistance to fluoroquinolones and quinolones (Guillard et al., 2015).
The high prevalence of antibiotic resistance was echoed in other studies, including Opintan et al. 2015), Feglo and Adu-Sarkodie (2016), pointing to widespread antibiotic resistance in the bacterium, which has consequently become a serious public health problem. The species exhibits high resistance to broad-spectrum β-lactam antibiotics such as carbapenems via plasmid-encoded ESBLs and carbapenemases, including KPC, VIM, MBLS and OXA types (Castanheira et al., 2011; Deshpande et al., 2014).
Challenges to resistance control strategies
Enterobacter spp have EefABC and AcrAB-TolC efflux genes that export antibiotics from cytosolic or periplasmic region to extracellular environment, which contribute to resistance to fluoroquinolones and aminoglycosides (Martins et al., 2010). Apart from colistin and tigecycline, few antibiotics are effective against these resistant strains due to the multi-drug resistant properties, and therefore there are few or no drugs in the 'pipeline' known to be able to effectively deal with its growing health crisis ( Bergen et al., 2015; In Ghana, information on Enterobacter spp resistance is scarce, however, few studies on antibiotic resistance prevalence, including that of Obeng-Nkrumah et al. 2013), which attempted to investigate the monitoring and evaluation of antibiotic resistance in a major teaching hospital, and Newman et al. 2011) in their research on antimicrobial resistance drugs in Ghana have indicated high resistance among the species to most of the first-line antibiotics used in the country, which is cause for concern.
These are major obstacles to the effective management and control of antibiotic resistance, especially in sub-Saharan African countries, including Ghana (Gandra, Merchant, & Laxminarayan, 2016; Ndihokubwayo et al., 2010; WHO, 2014). Selection pressure due to the misuse of antibiotics significantly contributes to the emergence of bacterial resistance (Michael et al., 2014).
AIM OF STUDY
SPECIFIC OBJECTIVES
STUDY DESIGN AND METHODOLOGY
Study design
Ethical approval was obtained from the Joint Committee of Human Research Publications and Ethics, School of Medical Sciences, Kwame Nkrumah University of Technology, Research and Development Unit of the Hospital Administration (ref: CHRPE/AP/015/15) and the Biomedical Research Ethics Committee of the University of Kwa-Zulu Natal (ref: BE 494/14). Voluntary informed consent was obtained from all participants, in writing or in the form of a thumbprint. Parents or guardians of minors gave consent after explaining the procedure and purpose of the study.
Methodology
Govinden, as co-supervisor, supervised the laboratory analysis and undertook critical revision of the manuscript. Owusu-Ofori as co-supervisor, co-conceived the study, supervised sampling and preliminary laboratory work, and undertook critical revision of the manuscript. Govinden, as co-supervisor, co-conceived the study, facilitated analysis of whole genome sequencing results, and undertook critical revision of the manuscript.
Sundsfjord, contributed to data analyzes and writing process, ensuring the quality of the final manuscript. Essack, as principal investigator, co-conceptualized the study and undertook critical revision of the manuscript. Global phylogenic investigation indicated that two of the isolates (P26-75 and P26-81) were of the same sequence type (ST101) as the main group of carbapenemase-producing K.
Characterization of the CTX-M-15 encoding gene in Klebsiella pneumoniae strains from the Barcelona metropolitan area: plasmid diversity and chromosomal integration. Delineating the clones and MGEs encoding the resistance genes in this pathogen is critical to contain their proliferation. The genomic DNA libraries were generated using the Nextera® kit (Illumina), followed by sequencing on an Illumina MiSeq platform at the Genomics Resource Center of UiT – The Arctic University of Norway.
Further insights into the blaDIM-1 and blaIMP-34-like coding regions of the P26-85 chromosome were obtained by alignment with the corresponding regions of P. Furthermore, the alignments revealed the presence of two genomic islands (green arrows) containing , revealed each of the integron structures. A linkage of both the blaDIM-1 and blaIMP-34-like genes to type1 integron structures, including the intI1 integrase encoding gene, was found by BLAST analyzes of the assembled contigs.
Putative integron structures (white arrows) with the two genomic islands containing each of the integron structures (green arrows) at the top of the figure. Positive association analyzes of blaDIM-1 and blaIMP-34 showed an association of the blaDIM-1 and blaIMP-34-like type 1 integron structure with the integrase-encoding gene intI1.
Conclusion
Multiple and diverse mutations were detected in quinolone resistance-determining regions of gyrA , gyrB and parC genes among K .
Limitations
Recommendation
Emergence of carbapenem resistance due to novel insertion sequence IS Pa 8 in Pseudomonas aeruginosa. Antibiotic resistance profile of non-extended spectrum β-lactamase producing Escherichia coli and Klebsiella pneumoniae in Accra. Phenotypic Determination and Antimicrobial Resistance Profile of Extended Spectrum β-Lactamases in Escherichia coli and Klebsiella pneumoniae in Accra, Ghana.
Carbapenem resistance in Pseudomonas aeruginosa and Acinetobacter baumannii in nosocomial settings in Latin America. MexY-promoted aminoglycoside resistance in Pseudomonas aeruginosa: involvement of a putative proximal binding pocket in aminoglycoside recognition. Structure and function of OprD protein in Pseudomonas aeruginosa: from antibiotic resistance to novel therapies.
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First detection of OXA-10 extended-spectrum β-lactamases and the occurrence of mexR and nfxB in clinical isolates of Pseudomonas aeruginosa from Nigeria. Insertion sequence ISRP10 inactivation of the oprD gene in imipenem-resistant Pseudomonas aeruginosa clinical isolates. Antimicrobial resistance pattern and their beta-lactamase-encoding genes among Pseudomonas aeruginosa strains isolated from cancer patients.
