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Enterococci are among the most common opportunistic hospital pathogens. This study used whole-genome sequencing (WGS) and bioinformatics to determine the antibiotic resistome, mobile genetic elements, clone and phylogenetic relationship of Enterococcus faecalis isolated from hospital environments in South Africa. This study was carried out from September to November 2017. Isolates were recovered from 11 frequently touched sites by patients and healthcare workers in different wards at 4 levels of healthcare (A, B, C, and D) in Durban, South Africa. Out of the 245 identified E. faecalis isolates, 38 isolates underwent whole-genome sequencing (WGS) on the Illumina MiSeq platform, following microbial identification and antibiotic susceptibility tests. The tet(M) (31/38, 82%) and erm(C) (16/38, 42%) genes were the most common antibiotic-resistant genes found in isolates originating from different hospital environments which corroborated with their antibiotic resistance phenotypes. The isolates harboured mobile genetic elements consisting of plasmids ( n  = 11) and prophages ( n  = 14) that were mostly clone-specific. Of note, a large number of insertion sequence (IS) families were found on the IS3 (55%), IS5 (42%), IS1595 (40%), and Tn3 transposons the most predominant. Microbial typing using WGS data revealed 15 clones with 6 major sequence types (ST) belonging to ST16 ( n  = 7), ST40 ( n  = 6), ST21 ( n  = 5), ST126 ( n  = 3), ST23 ( n  = 3), and ST386 ( n  = 3). Phylogenomic analysis showed that the major clones were mostly conserved within specific hospital environments. However, further metadata insights revealed the complex intraclonal spread of these E. faecalis major clones between the sampling sites within each specific hospital setting. The results of these genomic analyses will offer insights into antibiotic-resistant E. faecalis in hospital environments relevant to the design of optimal infection prevention strategies in hospital settings., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2023 Christiana O. Shobo et al.)

The work aims to investigate biofilm formation and biofilm/adhesion-encoding genes in coagulase-negative staphylococci (CoNS) species recovered from blood culture isolates. Eighty-nine clinical CoNS were confirmed using the VITEK 2 system, and antibiotic susceptibility testing of isolates was conducted using the Kirby-Bauer disk diffusion method against a panel of 20 antibiotics. Isolates were qualitatively screened using the Congo red agar medium. Quantitative assays were performed on microtiter plates, where the absorbances of the solubilised biofilms were recorded as optical densities and quantified. In all, 12.4% of the isolates were strong biofilm formers, 68.5% had moderate biofilm capacity, and 17.9% showed weak capacity. A subset of 18 isolates, mainly methicillin-resistant S. epidermidis , were investigated for adherence-related genes using whole-genome sequencing and bioinformatics analysis. The highest antibiotic resistance rates for strongly adherent isolates were observed against penicillin (100%) and cefoxitin (81.8%), but the isolates showed no resistance to linezolid (0.0%) and tigecycline (0.0%). The ica ABC genes involved in biofilm formation were detected in 50% of the screened isolates. Other adherence-related genes, including autolysin gene atl (88.8%), elastin binding protein gene ebp (94.4%), cell wall-associated fibronectin-binding protein gene ebh (66.7%), clumping factor A gene clf A (5.5%), and pili gene ebp C (22.2%) were also found. The insertion sequence IS256, involved in biofilm formation, was found in 10/18 (55.5%) screened isolates. We demonstrate a high prevalence of biofilm-forming coagulase-negative staphylococci associated with various resistance phenotypes and a substantial agreement between the possession of biofilm-associated genes and the biofilm phenotype.

Bacteria that cause life-threatening illnesses in humans are also capable of contaminating hospital surfaces, thus pose as a potential source of infection. This study aimed to investigate the prevalence, genetic diversity, virulence, and antibiotic resistance profile of Klebsiella pneumoniae in South Africa. In a nonoutbreak setting involving four public hospitals, 777 samples were collected in three different wards from 11 different sites. Phenotypic and genotypic methods were used for isolation and identification. The Kirby-Bauer disk-diffusion method was used to examine antibiotic resistance followed by the combination disk method to characterize extended-spectrum β-lactamases (ESBLs). Antibiotic resistance and virulence genes were screened using PCR and clonality was investigated using enterobacterial repetitive intergenic consensus (ERIC)-PCR. Seventy-five (10%) K. pneumoniae isolates were recovered. These isolates were obtained from all four hospitals and all three wards involved. However, only six frequently touched surfaces were contaminated. Thirty (40%) isolates were characterized as ESBLs showing high resistance to antibiotics and mostly harboring the bla CTX-M group one gene. Virulence genes were highly prevalent among all the isolates. ERIC-PCR showed that the isolates recovered from different sites within the same hospital were genetically similar. The study highlighted that K. pneumoniae can contaminate various surfaces and this persistence allows for the dissemination of bacteria within the hospital environment. The information from this study can assist hospitals to evaluate and improve current infection prevention and control interventions in place.

This study aimed to assess the molecular dissemination of Bacillus species in public hospitals in South Africa. The study conducted over 3 months during 2017 involved representative samples obtained from three wards (general ward, intensive care unit, and pediatric unit) from four public hospitals denoted as A (Central), B (Tertiary), C (Regional), and D (District). Swabs collected from 11 distinct hospital surfaces were screened using selective media, biochemical testing, and molecular methods. Overall, 17% (135/777) isolates were identified with a prevalence of 24% (32/135) for central, 33% (45/135) for tertiary, 27% (36/135) for regional, and 16% (22/135) for district hospital. Bacillus species were further confirmed to belong to Bacillus cereus (129/135; 96%) and Bacillus subtilis (6/135; 4%). Prevalence was similar across the wards, averaging 33.3% (45/135). The highest prevalence of Bacillus isolates was found on the drip stands (11.8%), sink (11.8%), ward phone (11.5%), and nurses' tables (10.3%). Minimum inhibitory concentration analyses revealed high resistance to β-lactams, fluoroquinolones, and tetracyclines. The most common resistance genes detected were ermB (56%) and t etM (5%). Enterotoxin virulence genes hblA (77%) and hblD (88%) associated with the diarrheal syndrome were most detected; however, no ces genes (cereulide toxin) for emetic syndrome was found. The enterobacterial repetitive intergenic consensus PCR revealed considerable diversity at the different levels of health care, although the clonal spread of strains between the sites/wards within each specific hospital was revealed. The study highlighted the dissemination of drug-resistant Bacillus spp. in public hospital environments and calls for the design of optimal strategies to curb their spread.

Background: This study determined the prevalence and antibiotic susceptibility profiles of Staphylococcus aureus isolated from selected critical control points (farm, transport, abattoir, and retail product) in an intensive poultry production system in the uMgungundlovu District, South Africa, using the "farm to fork" approach. Materials and Methods: Three hundred eighty-four samples from poultry and poultry products were examined across the "farm to fork" continuum for S. aureus using selective media, biochemical tests, and API Staph kit and confirmed by polymerase chain reaction identification of the nuc gene. Antibiotic susceptibility testing of the isolates was determined by the Kirby-Bauer disc diffusion method to 19 antimicrobials and to vancomycin by the broth microdilution technique. Results: The overall prevalence rate of S. aureus was 31.25% ( n  = 120/384), distributed across the continuum: farm site (40), transport (15), abattoir (30), and retail point (35). The isolates were resistant to tetracycline (61.67%), penicillin G (55.83%), erythromycin (54.17%), clindamycin (43.33%), doxycycline (36.67%), ampicillin (34.17%), moxifloxacin (30.83%), amikacin (30.83%), trimethoprim-sulfamethoxazole (30.00%), and levofloxacin (23.33%). A 100% susceptibility to tigecycline, teicoplanin, vancomycin, nitrofurantoin, chloramphenicol, and linezolid was observed in all isolates. The rate of multidrug resistance and the multiple antibiotic resistance index of the strains were 39.17% and 0.23%, respectively. The isolates showed similar patterns of resistance to commonly used growth promoters and antibiotics in veterinary and human medicine belonging to the same class. Conclusion: It is evident that the different antibiotics and growth promoters used in poultry production are exerting selection pressure for the emergence and co-selection of antibiotic-resistant bacteria in the production system, necessitating efficient antibiotic stewardship guidelines to streamline their use.

Carbapenem-resistant Enterobacteriaceae (CREs)-mediated infections remain a huge public health concern. CREs produce enzymes such as metallo-β-lactamases (MBLs), which inactivate β-lactam antibiotics. Hence, developing efficient molecules capable of inhibiting these enzymes remains a way forward to overcoming this phenomenon. In this study, we demonstrate that pyridyl moieties favor the inhibitory activity of cyclic metal-chelating agents through in vitro screening, molecular modeling, and docking assays. Di-(2-picolyl) amine and tris-(2-picolyl) amine exhibited great efficacy against different types of MBLs and strong binding affinity for NDM-1, whereas 2-picolyl amine did not show activity at a concentration of 64 mg/L in combination with meropenem; it further showed the lowest binding affinity from computational molecular analysis, commensurating with the in vitro screening assays. The findings revealed that the pyridyl group plays a vital role in the inhibitory activity of the tested molecules against CREs and should be exploited as potential MBL inhibitors.

Aims: We hypothesized and confirmed that tannic acid (TA) reverses carbapenem resistance by inhibiting carbapenemases in class A and B carbapenemase-producing Enterobacteriaceae., Methods and Results: Minimum inhibitory concentrations of carbapenems in the presence and absence of TA and other efflux pump inhibitors, TA-carbapenemases inhibition assays and computational studies showed that TA had the greatest effect on metallo-β-lactamases (MBLs) followed by class A serine-β-lactamases (SBLs). TA completely reversed the MICs of MBL producers from between 32 and ≥512 mg l -1 to susceptible values (<4 mg l -1 ) while substantially reducing the MICs of SBLs from between 16 and >512 mg l -1 to <4 to 16 mg l -1 . Tolerable cytotoxic effect was observed for the concentrations tested (8-1024 mg l -1 ). TA inhibited enzymes with a marked difference of ≈50% inhibition (IC 50 ) for NDM-1 (270 μmol l -1 ) and KPC-2 (15  μmol l -1 )., Conclusion: TA inhibited both MBLs and SBLs by targeting their hydrophobic sites. Moreover, TA had a stronger binding affinity for MBLs than SBLs as the MBLs, specifically VIM-1 (-43·7220 ± 0·4513 kcal mol -1 ) and NDM-1(-44·2329 ± 0·3806 kcal mol -1 ), interact with a larger number of their catalytic active-site residues than that of OXA-48 (-22·5275 ±  0·1300 kcal mol -1 ) and KPC-2 (-22·1164 ± 0·0111 kcal mol -1 )., Significance and Impact of the Study: Tannic acid or its analogues could be developed into carbapenemase-inhibiting adjuvants to restore carbapenem activity in CRE infections, save many lives and reduce healthcare associated costs., (© 2018 The Society for Applied Microbiology.)

Metallo-β-lactamase (MBL)-producing Enterobacteriaceae are of grave clinical concern, particularly as there are no metallo-β-lactamase inhibitors approved for clinical use. The discovery and development of MBL inhibitors to restore the efficacy of available β-lactams are thus imperative. We investigated a zinc-chelating moiety, 1,4,7-triazacyclononane (TACN), for its inhibitory activity against clinical carbapenem-resistant Enterobacteriaceae MICs, minimum bactericidal concentrations (MBCs), the serum effect, fractional inhibitory concentration indexes, and time-kill kinetics were determined using broth microdilution techniques according to Clinical and Laboratory Standards Institute (CSLI) guidelines. Enzyme kinetic parameters and the cytotoxic effects of TACN were determined using spectrophotometric assays. The interactions of the enzyme-TACN complex were investigated by computational studies. Meropenem regained its activity against carbapenemase-producing Enterobacteriaceae , with the MIC decreasing from between 8 and 64 mg/liter to 0.03 mg/liter in the presence of TACN. The TACN-meropenem combination showed bactericidal effects with an MBC/MIC ratio of ≤4, and synergistic activity was observed. Human serum effects on the MICs were insignificant, and TACN was found to be noncytotoxic at concentrations above the MIC values. Computational studies predicted that TACN inhibits MBLs by targeting their catalytic active-site pockets. This was supported by its inhibition constant ( K i ), which was 0.044 μM, and its inactivation constant ( K inact ), which was 0.0406 min -1 , demonstrating that TACN inhibits MBLs efficiently and holds promise as a potential inhibitor. IMPORTANCE Carbapenem-resistant Enterobacteriaceae (CRE)-mediated infections remain a significant public health concern and have been reported to be critical in the World Health Organization's priority pathogens list for the research and development of new antibiotics. CRE produce enzymes, such as metallo-β-lactamases (MBLs), which inactivate β-lactam antibiotics. Combination therapies involving a β-lactam antibiotic and a β-lactamase inhibitor remain a major treatment option for infections caused by β-lactamase-producing organisms. Currently, no MBL inhibitor-β-lactam combination therapy is clinically available for MBL-positive bacterial infections. Hence, developing efficient molecules capable of inhibiting these enzymes could be a promising way to overcome this phenomenon. TACN played a significant role in the inhibitory activity of the tested molecules against CREs by potentiating the activity of carbapenem. This study demonstrates that TACN inhibits MBLs efficiently and holds promises as a potential MBL inhibitor to help curb the global health threat posed by MBL-producing CREs., (Copyright © 2019 American Society for Microbiology.)

Introduction: There is a dearth of surveillance data on clinical Campylobacter in South Africa, particularly in the private healthcare environment. We investigated the prevalence of resistance to first-line antibiotics used to treat campylobacterioses in clinical Campylobacter isolates from a private pathology laboratory., Methodology: Identification of the Campylobacter specific genes were confirmed by PCR. Minimum inhibitory concentrations were determined using the broth micro-dilution method against macrolides (erythromycin, azithromycin), fluoroquinolones (ciprofloxacin, gatifloxacin) and tetracycline., Results: Seventy-two Campylobacter isolates were identified by PCR, with 54 (75%) being classified as C. jejuni and 18 (25%) as C. coli. Of these, 11 (20.4%) C. jejuni and six (33.3%) C. coli strains were resistant to ciprofloxacin and three (7.41%) C. jejuni and three (16.7%) C. coli strains were resistant to gatifloxacin. The number of C. jejuni strains resistant to erythromycin and azithromycin was 17 (31.5%) and 36 (50%) respectively, while the resistance of C. coli strains to erythromycin and azithromycin were seven (38.9%) and 14 (77.8%) respectively. Resistance to tetracycline was detected in 10 (55.6%) C. coli and 14 (25.9%) C. jejuni strains., Conclusion: In the light of these resistant profiles, the lack of a South African Campylobacter surveillance program is of concern. Relatively high prevalence of resistance in clinical isolates of C. jejuni and C. coli to the fluoroquinolones, macrolides and tetracycline used in first line treatment is of great concern. The efficacy treating human campylobacteriosis should thus be revisited.

A literature review was undertaken to ascertain the molecular basis for tigecycline and colistin resistance mechanisms and the experimental basis for the detection and delineation of this resistance particularly in carbapenemase-producing Gram-negative bacteria. Pubmed, Google Scholar and Science Direct were searched with the keywords colistin, tigecycline, resistance mechanisms and detection methods. Trans-complementation and comparative MIC studies, mass spectrometry, chromatography, spectrofluorometry, PCR, qRT-PCR and whole genome sequencing (WGS) were commonly used to determine tigecycline and colistin resistance mechanisms, specifically modifications in the structural and regulatory efflux (acrAB, OqxAB, kpgABC adeABC-FGH-IJK, mexAB-XY-oprJM and soxS, rarA robA, ramRAB marRABC, adeLRS, mexRZ and nfxb) and lipid A (pmrHFIJFKLM, lpxA, lpxC lpxD and mgrB, pmrAB, phoPQ,) genes respectively. Mutations in the ribosomal 16S rRNA operon rrnBC, also yielded resistance to tigecycline through target site modifications. The mcr-1 gene conferring resistance to colistin was identified via WGS, trans-complementation and a murine thigh infection model studies. Common detection methods are mainly antibiotic sensitivity testing with broth microdilution while molecular identification tools are mostly PCR and WGS. Spectrofluorometry, MALDI-TOF MS, micro-array and real-time multiplex PCR hold much promise for the future as new detection tools., (© 2016 The Society for Applied Microbiology.)

Fluoroquinolones are broad-spectrum antibiotics with efficacy against a wide range of pathogenic microbes associated with respiratory and meningeal infections. The potential toxicity of this class of chemical agents is a source of major concern and is becoming a global issue. The aim of this study was to develop a method for the brain distribution and the pharmacokinetic profile of gatifloxacin in healthy Sprague-Dawley rats, via Multicenter matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) and quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS). We developed a sensitive LC-MS/MS method to quantify gatifloxacin in plasma, lung, and brain homogenates. A pharmacokinetic profile was observed where there is a double peak pattern; a sharp initial increase in the concentration soon after dosing followed by a steady decline until another increase in concentration after a longer period post dosing in all three biological samples was observed. The imaging results showed the drug gradually entering the brain via the blood brain barrier and into the cortical regions from 15 to 240 min post dose. As time elapses, the drug leaves the brain following the same path as it followed on its entry and finally concentrates at the cortex. Copyright © 2015 John Wiley & Sons, Ltd., (Copyright © 2015 John Wiley & Sons, Ltd.)

Tigecycline (TIG), a derivative of minocycline, is the first in the novel class of glycylcyclines and is currently indicated for the treatment of complicated skin structure and intra-abdominal infections. A selective, accurate and reversed-phase high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed for the determination of TIG in rat brain tissues. Sample preparation was based on protein precipitation and solid phase extraction using Supel-Select HLB (30 mg/1 mL) cartridges. The samples were separated on a YMC Triart C18 column (150 mm x 3.0 mm. 3.0 µm) using gradient elution. Positive electrospray ionization (ESI+) was used for the detection mechanism with the multiple reaction monitoring (MRM) mode. The method was validated over the concentration range of 150-1200 ng/mL for rat brain tissue. The precision and accuracy for all brain analyses were within the acceptable limit. The mean extraction recovery in rat brain was 83.6%. This validated method was successfully applied to a pharmacokinetic study in female Sprague Dawley rats, which were given a dose of 25 mg/kg TIG intraperitoneally at various time-points. Copyright © 2015 John Wiley & Sons, Ltd., (Copyright © 2015 John Wiley & Sons, Ltd.)

Objectives: Due to the lack of information on the plasmid content of MRSA strains in South Africa (SA), this study investigated the resistance and virulence mechanisms of 27 clinical isolates from the private health care sector over a period of 3 months., Methods: Plasmids were extracted and the presence of MRSA confirmed by the presence of mecA. The isolates were subjected to antimicrobial susceptibility testing and molecular characterization of common resistance encoding genes and frequently encountered virulence factors by PCR using plasmid DNA as the template. The genetic relatedness between the isolates was determined by pulsed field gel electrophoresis (PFGE)., Results: All isolates were plasmid positive, and displayed ampillicin, ciprofloxacin, gentamicin, rifampicin, tetracycline, erythromycin, and clindamycin resistance. They were all fully susceptible to daptomycin, linezolid, vancomycin, tigecycline and fusidic acid. Multidrug resistance (MDR) was found in 74.1% (20/27) of the MRSA isolates. The frequency of the resistance and virulence genes ranged from 100% to 0%. PFGE analysis revealed 10 pulsotypes, designated A-J, which showed correlation with resistance profile of the isolates in each group. Of note, 85.2% (23/27) of the isolates clustered into six major PFGE types giving an indication of similar circulating MRSA clones., Conclusions: This study highlights the genetic diversity and resistance mechanisms in MRSA strains from the private health sector in SA hence the need for implementing effective infection control programs., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Aims: This study aimed at investigating the use of metal chelators as potential metallo-β-lactamase inhibitors (MBL)., Methods and Results: The minimum inhibitory concentration (MIC) of meropenem was ascertained alone and in combination with various concentrations of macrocyclic (1,4,7- triazacyclononane-1-glutaric acid-4,7-diacetic acid = NODAGA) peptide derivatives and acyclic (N,N,N',N'-Tetrakis(2-pyridylmethyl)ethylenediamine = TPEN and di-(2-picolyl)amine = DPA) metal chelators using the broth microdilution method. MICs of meropenem against carbapenem-resistant enterobacteriaceae (CRE) producing MBLs were decreased to concentrations as low as 0·06 mg l(-1) in the presence of some metal chelators. TPEN at 4 and 8 mg l(-1) showed the best activity by decreasing meropenem MICs to 0·5 and 0·06 mg l(-1) , respectively, for some New Delhi Metallo-beta-lactamase (NDM) and Verona integron-encoded metallo-β-lactamase (VIM) -producing enterobacteriaceae. DPA at 8 and 16 mg l(-1) was also able to decrease meropenem MICs to 1 and 0·125 mg l(-1) , respectively, for these CREs. NODAGA peptide derivatives showed the least inhibition as 32 mg l(-1) was required for meropenem MICs to be decreased to 0·06 mg l(-1) against an NDM-1 producing isolate., Conclusion: The various metal chelators, TPEN, DPA and NODAGA peptide derivatives were able to inhibit the MBLs in decreasing order of activity, rendering CREs susceptible to meropenem., Significance and Impact of the Study: In the absence of new antibiotics, this study evaluated metal chelators as potential MBL inhibitors., (© 2016 The Society for Applied Microbiology.)

Campylobacter bacteria are important foodborne pathogens that cause acute diarrheal illness, and infection is often associated with contaminated poultry. In a blind observational study, the prevalence and resistance profiles of thermophilic Campylobacter strains collected from different poultry production systems were tested against the clinically used antibiotics ciprofloxacin, tetracycline, erythromycin, gentamicin, and streptomycin. Campylobacter strains were isolated from chickens in rural production systems, a free-range commercial facility, and industrially raised broiler and egg-laying chickens all situated in KwaZulu-Natal, South Africa. Isolates were collected from the chicken cecae and were identified with conventional methods and tested for antibiotic resistance with the Clinical and Laboratory Standards Institute agar dilution method. The prevalence of Campylobacter spp. isolates in chickens was 68% (56 samples) in rural production, 47% (140 samples) in commercial free-range broilers, 47% (133 samples) in industrial broilers, and 94% (34 samples) in industrial layer hens. Isolates from the rurally raised chickens showed significantly (P < 0.01) less resistance against ciprofloxacin (7.9%), erythromycin (0%), and tetracycline (21.6%) than those from commercially produced chickens. Isolates from the commercially raised chickens (free range and industrial) were highly resistant to tetracycline (98.9 to 100%). The incidence of gentamicin and streptomycin resistance was 1.6 and 11.5%, respectively, in commercial free-range broilers, 1.7 and 16.4%, respectively, in industrially raised broilers, and 12.9 and 40%, respectively, in industrially raised layers. It is possible that variations among the poultry production systems, including antimicrobial usage, result in differences in antibiotic resistance profiles in Campylobacter.

Objectives: Campylobacter jejuni isolated from broiler and layer chickens from registered abattoirs in KwaZulu-Natal, South Africa, were tested for their susceptibility to eight antibiotics., Methods: Using agar dilution, susceptibility to eight antibiotics was determined for C. jejuni recovered from the caeca., Results: A total of 155 isolates were collected of which 77 were identified as C. jejuni (broilers n = 56 and layers n = 21). Resistance was highest to tetracycline (broilers 98.2% and layers 100%) and ceftriaxone (broilers 96.4% and layers 100%). High susceptibility was found to ciprofloxacin (broilers 91% and layers 76%) and gentamicin (broilers 98% and layers 81%). Susceptibilities to each of the antibiotics for the broilers and layers, respectively, were: 50% and 57% for erythromycin, 45% and 24% for clarithromycin, 68% and 43% for ampicillin and 64% and 48% for nalidixic acid. Statistically significant differences were detected for the MIC(50) of gentamicin, ciprofloxacin and tetracycline between broilers and layers (P < 0.001) with the MIC(90) of gentamicin also of significant difference (P = 0.01). Multiresistance was detected in 23% and 43% of the isolates from broiler and layer chickens, respectively., Conclusions: Mass therapy procedures used in animal husbandry have a potential impact on antibiotic resistance development in C. jejuni.