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Introduction: Diarrhoeagenic Escherichia coli (DEC) persistently challenges public health in Africa, contributing substantially to the diarrhoeal disease burden. This systematic review and meta-analysis illuminate the distribution and antimicrobial resistance (AMR) patterns of DEC pathotypes across the continent., Methods: The review selectively focused on pathotype-specific studies reporting prevalence and/or AMR of human-derived DEC pathotypes from African nations, excluding data from extra-intestinal, animal, and environmental sources and studies focused on drug and mechanism experiments. Pertinent studies were retrieved from SCOPUS, PubMed, and EBSCOhost, processed with Covidence, and screened in alignment with PRISMA guidelines., Results: The reviewed studies were predominantly hospital-based (80%) and paediatric-focused (91%), with a meagre 4.4% documenting DEC outbreaks. Seven DEC pathotypes were discerned, with Enteroaggregative E. coli (EAEC) being notably prevalent (43%, 95% CI 30-55%) and Enteroinvasive E. coli (EIEC) least prevalent (24%, 95% CI 17-32%). Identified non-susceptibilities were noted against essential antibiotics including ciprofloxacin, ceftriaxone, and ampicillin, while instances of carbapenem and Extended-Spectrum ß-Lactamase (ESBL) resistance were scarce., Conclusion: Despite sporadic data on DEC prevalence and AMR in Africa, particularly in community settings, a palpable gap remains in real-time outbreak surveillance and comprehensive data documentation. Augmenting surveillance and embracing advancements in molecular/genomic characterisation techniques are crucial to precisely discerning DEC's actual impact and resistance continuum in Africa., (© 2024. The Author(s).)

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.)

Virulent Enterobacterale strains expressing serine and metallo-β-lactamases (MBL) genes have emerged responsible for conferring resistance to hard-to-treat infectious diseases. One strategy that exists is to develop β-lactamase inhibitors to counter this resistance. Currently, serine β-lactamase inhibitors (SBLIs) are in therapeutic use. However, an urgent global need for clinical metallo-β-lactamase inhibitors (MBLIs) has become dire. To address this problem, this study evaluated BP2, a novel beta-lactam-derived β-lactamase inhibitor, co-administered with meropenem. According to the antimicrobial susceptibility results, BP2 potentiates the synergistic activity of meropenem to a minimum inhibitory concentration (MIC) of ≤1 mg/L. In addition, BP2 is bactericidal over 24 h and safe to administer at the selected concentrations. Enzyme inhibition kinetics showed that BP2 had an apparent inhibitory constant (K i app ) of 35.3 µM and 30.9 µM against New Delhi Metallo-β-lactamase (NDM-1) and Verona Integron-encoded Metallo-β-lactamase (VIM-2), respectively. BP2 did not interact with glyoxylase II enzyme up to 500 µM, indicating specific (MBL) binding. In a murine infection model, BP2 co-administered with meropenem was efficacious, observed by the >3 log 10 reduction in K. pneumoniae NDM cfu/thigh. Given the promising pre-clinical results, BP2 is a suitable candidate for further research and development as an (MBLI).

β-lactams are the most prescribed class of antibiotics due to their potent, broad-spectrum antimicrobial activities. However, alarming rates of antimicrobial resistance now threaten the clinical relevance of these drugs, especially for the carbapenem-resistant Enterobacterales expressing metallo-β-lactamases (MBLs). Antimicrobial agents that specifically target these enzymes to restore the efficacy of last resort β-lactam drugs, that is, carbapenems, are therefore desperately needed. Herein, we present a cyclic zinc chelator covalently attached to a β-lactam scaffold (cephalosporin), that is, BP1. Observations from in vitro assays (with seven MBL expressing bacteria from different geographies) have indicated that BP1 restored the efficacy of meropenem to ≤ 0.5 mg/L, with sterilizing activity occurring from 8 h postinoculation. Furthermore, BP1 was nontoxic against human hepatocarcinoma cells (IC 50 > 1000 mg/L) and exhibited a potency of (K i app ) 24.8 and 97.4 μM against Verona integron-encoded MBL (VIM-2) and New Delhi metallo β-lactamase (NDM-1), respectively. There was no inhibition observed from BP1 with the human zinc-containing enzyme glyoxylase II up to 500 μM. Preliminary molecular docking of BP1 with NDM-1 and VIM-2 sheds light on BP1's mode of action. In Klebsiella pneumoniae NDM infected mice, BP1 coadministered with meropenem was efficacious in reducing the bacterial load by >3 log 10 units' postinfection. The findings herein propose a favorable therapeutic combination strategy that restores the activity of the carbapenem antibiotic class and complements the few MBL inhibitors under development, with the ultimate goal of curbing antimicrobial resistance.

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.

Poultry is a cheap source of animal protein and constituent of diets in Africa. Poultry can serve as a reservoir for Salmonella and cause food-borne infections in humans. This review describes Salmonella contamination of food, poultry, and the farming environment, antimicrobial resistance profiles, and serotypes of Salmonella , as well as the farming systems, antimicrobial use (AMU), hygiene, and husbandry conditions used to rear poultry in Africa. Using the PRISMA (preferred reporting items for systematic reviews and meta-analysis) guidelines, PubMed, Science Direct, and Web of Science databases were searched using a set of predefined keywords. Full-length research articles in English were examined for the period 2010-2020 and relevant information extracted for the narrative synthesis. Of the articles that met the inclusion criteria, 63.1% were conducted on farms and among households, while 36.9% were undertaken at government-controlled laboratories, which quarantine imported birds, processing plants, and retail outlets. The farming systems were intensive, semi-intensive, and extensive. AMU was described in 11.5% of the studies and varied within and across countries. Multidrug-resistant (MDR) Salmonella isolates were detected in 30 studies and the prevalence ranged from 12.1% in Zimbabwe to 100% in Egypt, Ethiopia, Nigeria, Senegal, and South Africa. A total of 226 different Salmonella serotypes were reported. Twenty-four (19.7%) of the studies reported food-borne Salmonella contamination in eggs, poultry, and poultry products at retail outlets and processing plants. The apparent extensive use of antimicrobials and circulation of MDR Salmonella isolates of various serotypes in Africa is a concern. It is important to implement stricter biosecurity measures on farms, regulate the use of antimicrobials and implement surveillance systems, in addition to food safety measures to monitor the quality of poultry and poultry products for human consumption.

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.

Background:  Diarrheagenic E. coli (DEC) strains are a major cause of diarrheal diseases in both developed and developing countries. Healthy asymptomatic animals may be reservoirs of zoonotic DEC, which may enter the food chain via the weak points in hygiene practices., Aim:  We investigated the prevalence of DEC along the pig production continuum from farm-to-fork., Methods:  A total of 417 samples were collected from specific points along the pig production system, that is, farm, transport, abattoir and food. E. coli was isolated and enumerated using Colilert. Ten isolates from each Quanti-tray were selected randomly and phenotypically identified using eosin methylene blue agar selective media. Real-time polymerase chain reaction (PCR) was used to confirm the species and to classify them into the various diarrheagenic pathotypes. Antimicrobial susceptibility was determined against a panel of 20 antibiotics using the Kirby-Bauer disk diffusion method and EUCAST guideline., Results:  The final sample size consisted of 1044 isolates, of which 45.40% (474/1044) were DEC and 73% (762/1044) were multidrug-resistant. Enteroinvasive E. coli (EIEC) was the most predominant DEC at all the sampling sites., Conclusion:  The presence of DEC in food animal production environments and food of animal origin could serve as reservoirs for transmitting these bacteria to humans, especially in occupationally exposed workers and via food. Adherence to good hygienic practices along the pig production continuum is essential for mitigating the risk of transmission and infection, and ensuring food safety.

The presence of the zoonotic pathogen Salmonella in the food supply chain poses a serious public health threat. This study describes the prevalence, susceptibility profiles, virulence patterns, and clonality of Salmonella from a poultry flock monitored over six weeks, using the farm-to-fork approach. Salmonella was isolated using selective media and confirmed to the genus and species level by real-time polymerase chain reaction (RT-PCR) of the invA and iroB genes, respectively. Antimicrobial susceptibility profiles were determined using Vitek-2 and the Kirby-Bauer disk diffusion method against a panel of 21 antibiotics recommended by the World Health Organisation Advisory Group on Integrated Surveillance of Antimicrobial Resistance (WHO-AGISAR). Selected virulence genes were identified by conventional PCR, and clonality was determined using enterobacterial repetitive intergenic consensus PCR (ERIC-PCR). Salmonella was present in 32.1% of the samples: on the farm (30.9%), at the abattoir (0.6%), and during house decontamination (0.6%). A total of 210 isolates contained the invA and iroB genes. Litter, faeces, and carcass rinsate isolates were classified as resistant to cefuroxime (45.2%), cefoxitin (1.9%), chloramphenicol (1.9%), nitrofurantoin (0.4%), pefloxacin (11.4%), and azithromycin (11%). Multidrug resistance (MDR) was observed among 3.8% of the isolates. All wastewater and 72.4% of carcass rinsate isolates were fully susceptible. All isolates harboured the misL, orfL, pipD, stn, spiC, hilA , and sopB virulence genes, while pefA , spvA , spvB , and spvC were absent. In addition, fliC was only present among the wastewater isolates. Various ERIC-PCR patterns were observed throughout the continuum with different subtypes, indicating the unrelated spread of Salmonella . This study concluded that poultry and the poultry environment serve as reservoirs for resistant and pathogenic Salmonella . However, there was no evidence of transmission along the farm-to-fork continuum., Competing Interests: Professor Sabiha Y. Essack is the chairperson of the Global Respiratory Infection Partnership and a member of the Global Hygiene Council, both sponsored by unconditional educational grants from Reckitt, UK. All other authors declare that they have no conflicts of interest., (Copyright © 2022 Melissa A. Ramtahal et al.)

Aims: Enterococci are implicated in hospital-acquired infections and show high tenacity on inanimate objects in the hospital environment. This study investigated the prevalence of Enterococcus spp. in selected wards in public hospitals at four levels of healthcare from a district in KwaZulu-Natal, South Africa., Methods and Results: Swabs were collected from frequently touched areas in the paediatric wards and intensive care units (ICUs). Presumptive Enterococcus spp. were isolated and confirmed to genus and species levels, followed by Kirby-Bauer disk diffusion against 14 antibiotics. The results showed that enterococci were recovered from all 11 surfaces tested with the highest contamination rate observed on occupied beds and mops used to clean floors. A total number of 295 Enterococcus was identified. Polymerase chain reaction identified Enterococcus faecalis 83.1% (245/295) and Enterococcus faecium 12.9% (38/295), while whole genome sequencing identified Enterococcus gallinarum 2.0% (6/295) and Enterococcus casseliflavus 2.0% (6/295). Significant prevalence was observed in paediatric wards 64.1% (189/295) compared with the ICUs 35.9% (106/295), p < 0.05, in central, regional and district hospitals. Collectively, 82.0% (242/295) of enterococcal isolates were multidrug resistant, and 80 different antibiograms were observed. The most prominent antibiogram for E. faecium was CIP-RIF-NIT-TET-ERY and for E. faecalis CIP-TET-ERY., Conclusion: E. faecalis was the most frequent enterococcal species isolated in all the hospitals investigated and correlates with studies conducted elsewhere. A substantially greater number of isolates were recovered from the paediatric wards compared with ICUs, and thus improved standards should be developed for infection control practices. It is suggested that the elevated use of antibiotics contributed to the increased nonsusceptible isolates observed from ICUs. This study highlighted the high recovery rate of enterococci in the hospital environment even in a nonoutbreak setting., Significance and Impact of the Study: Enterocci had a high prevalence rate on the surfaces within the hospitals studied. This study gives an insight into the possible roles all healthcare staff may play in infection control intervention, including proper handling of hospital cleaning equipment and lack of knowledge about the potential for bacteria dissemination., (© 2021 The Society for Applied Microbiology.)

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.

The hospital environment acts as a reservoir in the transmission of pathogens, such as MRSA, which may cause hospital-acquired infections. This study aimed to ascertain the prevalence, genetic relatedness, antibiotic resistance, and virulence profile of MRSA on some frequently touched hospital sites in South Africa. A total of 777 swabs were randomly collected from 11 frequently touched sites in the hospital environment of three wards of four public hospitals in the KwaZulu-Natal province of South Africa. Isolation of S. aureus and confirmation were done using genotypic and phenotypic methods. Antibiotic susceptibility testing was performed using the Kirby-Bauer disk-diffusion method. MRSA isolates were determined by the presence of the mecA gene. Virulence and resistance genes were detected using a standard monoplex PCR assay. ERIC-PCR was conducted to evaluate the genetic relatedness. An overall prevalence of 12.7% for S. aureus isolates was obtained. Out of these, 89.9% (89/99) were confirmed to be MRSA. The sites with the highest prevalence were the occupied beds (16.2% (16/99)), unoccupied beds (16.2% (16/99)), patient files (14.1% (14/99)), ward phones (13.1% (13/99)), and nurses' tables (14.1% (14/99)). The virulence genes with the highest observed frequency were hld (87 (87.9%)) and LukS/F-PV (53 (53.5%)). The resistance genes with the highest frequency were the tetM and tetK genes detected in 60 (60.6%) and 57 (57.6%) isolates, respectively. The ERIC-PCR results obtained indicated a high level of genetic diversity; however, intraclonal (within a hospital) and interclonal (between hospitals) clusters of MRSA were observed. The study showed that MRSA can contaminate various surfaces, and this persistence allows for the dissemination of bacteria within the hospital environment. This highlights the need for improved infection prevention and control (IPC) strategies in public hospitals in the country to curb their potential transmission risks., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2021 Siyethaba Mkhize et al.)

Staphylococcus epidermidis has become an important nosocomial pathogen. Multidrug resistance makes S. epidermidis infections difficult to treat. The study aims to describe the genomic characteristics of methicillin-resistant S. epidermidis (MRSE) isolated from clinical sources, to comprehend the genetic basis of antibiotic resistance, virulence, and potential pathogenicity. Sixteen MRSE underwent whole-genome sequencing, and bioinformatics analyses were carried out to ascertain their resistome, virulome, mobilome, clonality, and phylogenomic relationships. In all, 75% of isolates displayed multidrug resistance and were associated with the carriage of multiple resistance genes including mecA , blaZ , tet(K) , erm(A) , erm(B) , erm(C) , dfrG , aac(6')-aph(2'') , and cat(pC221) conferring resistance to β-lactams, tetracyclines, macrolide-lincosamide-streptogramin B, aminoglycosides, and phenicols, which were located on both plasmids and chromosomes. Their virulence profiles were evidenced by the presence of genes involved in adherence/biofilm formation ( icaA , icaB , icaC , atl , ebh , and ebp ), immune evasion ( adsA , capC , and manA ), and antiphagocytosis ( rmlC , cdsA , and A ). The community-acquired SCC mec type IV was the most common SCC mec type. The CoNS belonged to seven multilocus sequence types (MLSTs) and carried a diversity of mobile genetic elements such as phages, insertion sequences, and plasmids. The bacterial anti-phage defense systems clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR-Cas) immunity phage system and restriction-modification system (R-M system) and the arginine catabolic mobile element (ACME) involved in immune evasion and transport of virulence genes were also found. The insertion sequence, IS256, linked with virulence, was found in 56.3% of isolates. Generally, the isolates clustered according to STs, with some similarity but also considerable variability within isolates. Whole-genome sequencing and bioinformatics analysis provide insights into the likely pathogenicity and antibiotic resistance of S. epidermidis , necessitating surveillance of this emerging pathogen., Competing Interests: SE is the chairperson of the Global Respiratory Infection Partnership and member of the Global Hygiene Council, both sponsored by unrestricted educational grants from Reckitt and Benckiser (Pty.) UK. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Asante, Hetsa, Amoako, Abia, Bester and Essack.)

Microbial infections are a major public health concern. Antimicrobial peptides (AMPs) have been demonstrated to be a plausible alternative to the current arsenal of drugs that has become inefficient due to multidrug resistance. Herein we describe a new AMP family, namely the super-cationic peptide dendrimers (SCPDs). Although all members of the series exert some antibacterial activity, we propose that special attention should be given to (KLK) 2 KLLKLL-NH 2 (G1KLK-L2KL2), which shows selectivity for Gram-negative bacteria and virtually no cytotoxicity in HepG2 and HEK293. These results reinforce the validity of the SCPD family as a valuable class of AMP and support G1KLK-L2KL2 as a strong lead candidate for the future development of an antibacterial agent against Gram-negative bacteria.

Foodborne pathogens, including antibiotic-resistant species, constitute a severe menace to food safety globally, especially food animals. Identifying points of concern that need immediate mitigation measures to prevent these bacteria from reaching households requires a broad understanding of these pathogens' spread along the food production chain. We investigated the distribution, antibiotic susceptibility, molecular characterization and clonality of Enterococcus spp. in an intensive pig production continuum in South Africa, using the farm-to-fork approach. Enterococcus spp. were isolated from 452 samples obtained along the pig farm-to-fork continuum (farm, transport, abattoir, and retail meat) using the IDEXX Enterolert ® /Quanti-Tray ® 2000 system. Pure colonies were obtained on selective media and confirmed by real-time PCR, targeting genus- and species-specific genes. The susceptibility to antibiotics was determined by the Kirby-Bauer disk diffusion method against 16 antibiotics recommended by the WHO-AGISAR using EUCAST guidelines. Selected antibiotic resistance and virulence genes were detected by real-time PCR. Clonal relatedness between isolates across the continuum was evaluated by REP-PCR. A total of 284 isolates, consisting of 79.2% E. faecalis , 6.7% E. faecium , 2.5% E. casseliflavus , 0.4% E. gallinarum , and 11.2% other Enterococcus spp., were collected along the farm-to-fork continuum. The isolates were most resistant to sulfamethoxazole-trimethoprim (78.8%) and least resistant to levofloxacin (5.6%). No resistance was observed to vancomycin, teicoplanin, tigecycline and linezolid. E. faecium displayed 44.4% resistance to quinupristin-dalfopristin. Also, 78% of the isolates were multidrug-resistant. Phenotypic resistance to tetracycline, aminoglycosides, and macrolides was corroborated by the presence of the tet M, aph(3')-IIIa , and erm B genes in 99.1%, 96.1%, and 88.3% of the isolates, respectively. The most detected virulence gene was gel E. Clonality revealed that E . faecalis isolates belonged to diverse clones along the continuum with major REP-types, mainly isolates from the same sampling source but different sampling rounds (on the farm). E. faecium isolates revealed a less diverse profile. The results suggest that intensive pig farming could serve as a reservoir of antibiotic-resistant bacteria that could be transmitted to occupationally exposed workers via direct contact with animals or consumers through animal products/food. This highlights the need for more robust guidelines for antibiotic use in intensive farming practices and the necessity of including Enterococcus spp. as an indicator in antibiotic resistance surveillance systems in food animals.

Campylobacter spp. are among the leading foodborne pathogens, causing campylobacteriosis, a zoonotic infection that results in bacterial gastroenteritis and diarrheal disease in animals and humans. This study investigated the molecular epidemiology of antibiotic-resistant Campylobacter spp. isolated across the farm-to-fork-continuum in an intensive pig production system in South Africa. Following ethical approval, samples were collected over sixteen weeks from selected critical points (farm, transport, abattoir, and retail) using a farm-to-fork sampling approach according to WHO-AGISAR guidelines. Overall, 520 samples were investigated for the presence of Campylobacter spp., which were putatively identified using selective media with identity and speciation confirmed by polymerase chain reaction (PCR) of specific genes. Resistance profiles were ascertained by the Kirby-Bauer disk diffusion method. Antibiotic resistance and virulence genes were identified using PCR and DNA sequencing. Clonal relatedness was determined using ERIC-PCR. Altogether, 378/520 (72.7%) samples were positive for Campylobacter spp., with Campylobacter coli being the predominant species (73.3%), followed by Campylobacter jejuni (17.7%); 8.9% of the isolates were classified as "other spp". Relatively high resistance was observed in C. coli and C. jejuni to erythromycin (89% and 99%), streptomycin (87% and 93%), tetracycline (82% and 96%), ampicillin (69% and 85%), and ciprofloxacin (53% and 67%), respectively. Multidrug resistance (MDR) was noted in 330 of the 378 (87.3%) isolates. The antibiotic resistance genes observed were tetO (74.6%), bla OXA- 61 (2.9%), and cmeB (11.1%), accounting for the resistance to tetracycline and ampicillin. The membrane efflux pump ( cmeB ), conferring resistance to multiple antibiotics, was also detected in most resistant isolates. Chromosomal mutations in gyrA (Thr-86-Ile) and 23S rRNA (A2075G and A2074C) genes, conferring quinolone and erythromycin resistance, respectively, were also found. Of the virulence genes tested, ciaB , dnaJ , pldA , cdtA , cdtB , cdtC , and cadF were detected in 48.6%, 61.1%, 17.4%, 67.4%, 19.3%, 51%, and 5% of all Campylobacter isolates, respectively. Clonal analysis revealed that isolates along the continuum were highly diverse, with isolates from the same sampling points belonging to the same major ERIC-types. The study showed relatively high resistance to antibiotics commonly used in intensive pig production in South Africa with some evidence, albeit minimal, of transmission across the farm-to-fork continuum. This, together with the virulence profiles present in Campylobacter spp., presents a challenge to food safety and a potential risk to human health, necessitating routine surveillance, antibiotic stewardship, and comprehensive biosecurity in intensive pig production.

Although Staphylococcus aureus is a major threat to the veterinary, agricultural, and public health sectors because of its zoonotic potential, studies on its molecular characterisation in intensive animal production are rare. We phenotypically and genotypically characterised antibiotic-resistant S. aureus in intensive pig production in South Africa, using the farm-to-fork approach. Samples ( n = 461) were collected from the farm, transport vehicles, and the abattoir using the World Health Organisation on Integrated Surveillance of Antimicrobial Resistance (WHO-AGISAR) sampling protocol. Bacteria were isolated using selective media and identified using biochemical tests and polymerase chain reaction (PCR). Phenotypic resistance was determined using the disk diffusion method. Selected resistance and virulence genes were investigated using PCR. Clonality among the isolates was determined using the repetitive element sequence-PCR. In all, 333 presumptive staphylococcal isolates were obtained, with 141/333 (42.3%) identified as staphylococci biochemically. Ninety-seven (97; 68.8%) were confirmed as S. aureus using PCR, 52.6% of which were identified as methicillin-resistant S. aureus (MRSA) through the mec A gene. All the 97 S. aureus isolates (100%) were resistant to at least one of the antibiotics tested, with the highest resistance observed against erythromycin and clindamycin (84.50% each), and the lowest observed against amikacin (2.10%); 82.47% (80/97) were multidrug-resistant with an average multiple antibiotic resistance index of 0.50. Most of the phenotypically resistant isolates carried at least one of the corresponding resistance genes tested, erm C being the most detected. hla was the most detected virulence gene (38.14%) and etb was the least (1.03%). Genetic fingerprinting revealed diverse MRSA isolates along the farm-to-fork continuum, the major REP types consisting of isolates from different sources suggesting a potential transmission along the continuum. Resistance to antibiotics used as growth promoters was evidenced by the high prevalence of MDR isolates with elevated multiple antibiotic resistance indices >0.2, specifically at the farm, indicating exposure to high antibiotic use environments, necessitating antibiotic stewardship and proper infection control measures in pig husbandry and intensive pig production.

Coagulase-negative staphylococci (CoNS) are increasingly associated with nosocomial infections, especially among the immunocompromised and those with invasive medical devices, posing a significant concern. We report on clinical multidrug-resistant CoNS from the uMgungundlovu District, KwaZulu-Natal Province, South Africa, as emerging pathogens. One hundred and thirty presumptive CoNS were obtained from blood cultures. Culture, biochemical tests, and the Staphaurex™ Latex Agglutination Test were used for the initial identification of CoNS isolates; confirmation and speciation were undertaken by the VITEK 2 system. Susceptibilities of isolates against a panel of 20 antibiotics were determined using the Kirby-Bauer disk diffusion method, and the multiple antibiotic resistance (MAR) indices of the isolates were determined. The polymerase chain reaction (PCR) was used to amplify the mec A gene to confirm methicillin resistance. Overall, 89/130 presumptive CoNS isolates were confirmed as CoNS by the VITEK 2 system. Of these, 68 (76.4%) isolates were putatively methicillin-resistant by the phenotypic cefoxitin screen test and 63 (92.6%) were mec A positive. Staphylococcus epidermidis (19.1%), S. hominis ssp. hominis (15.7%), and S. haemolyticus (16.9%) were the most common CoNS species. Isolates showed high percentage resistance against penicillin (100.0%), erythromycin (74.2%), and azithromycin (74.2%) while displaying high susceptibilities to linezolid (95.5%), gentamicin (95.5%), and tigecycline (94.4%). Multidrug resistance (MDR) was observed in 76.4% of isolates. MAR index calculation revealed 71.9% of isolates with MAR index >0.2 and 20.2% >0.5. Isolates with the highest MAR indices (0.7 and 0.8) were recovered from the neonatal intensive care unit. Fifty-one MDR antibiograms were observed. The high prevalence of methicillin resistance and multidrug resistance in several species of CoNS necessitates surveillance of this emerging pathogen, currently considered a contaminant of microbial cultures.

Antibiotic resistance profiles of Escherichia coli were investigated in an intensive pig production system in the uMgungundlovu District, South Africa, using the 'farm-to-fork' approach. Four hundred seventeen (417) samples were collected from pig and pig products at different points (farm, transport, and abattoir). E. coli was isolated and enumerated using the Colilert ® 18/Quanti-Tray ® 2000 system. Ten isolates from each Quanti-tray were selected randomly and putatively identified on eosin methylene blue agar. Real-time PCR targeting the uid A gene was used to confirm isolates to the genus level. The Kirby-Bauer disc diffusion method was used to determine the isolates' antibiotic susceptibility profiles against 20 antibiotics. A total of 1044 confirmed E. coli isolates were obtained across the three critical points in the food chain. Resistance was observed to all the antibiotics tested with the highest and lowest rates obtained against tetracycline (88.5%) and meropenem (0.2%), respectively. Resistance was also observed to chloramphenicol (71.4%), ampicillin (71.1%), trimethoprim-sulfamethoxazole (61.3%), amoxicillin-clavulanate (43.8%), cephalexin (34.3%), azithromycin (23.9%), nalidixic acid (22.1%), cefoxitin (21.1%), ceftriaxone (18.9%), ciprofloxacin (17.3%), cefotaxime (16.9%), gentamicin (15.5%), cefepime (13.8%), ceftazidime (9.8%), amikacin (3.4%), piperacillin-tazobactam (1.2%), tigecycline (0.9%), and imipenem (0.3%). Multidrug resistance (MDR) was observed in 71.2% of the resistant isolates with an overall multiple antibiotic resistance (MAR) index of 0.25, indicating exposure to high antibiotic use environments at the farm level. A high percentage of resistance was observed to growth promoters and antibiotics approved for veterinary medicine in South Africa. Of concern was resistance to critically important antibiotics for animal and human use and the watch and reserve categories of antibiotics. This could have adverse animal and human health consequences from a food safety perspective, necessitating efficient antibiotic stewardship and guidelines to streamline antibiotic use in the food-animal production chain.

The increased use of antibiotics in food animals has resulted in the selection of drug-resistant bacteria across the farm-to-fork continuum. This study aimed to investigate the molecular epidemiology of antibiotic-resistant Escherichia coli from intensively produced poultry in the uMgungundlovu District, KwaZulu-Natal, South Africa. Samples were collected weekly between August and September 2017 from hatching to final retail products. E. coli was isolated on eosin methylene blue agar, identified biochemically, and confirmed using polymerase chain reaction (PCR). Susceptibility to 19 antibiotics was ascertained by the Kirby-Bauer disc diffusion method. PCR was used to test for resistance genes. The clonal similarity was investigated using enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR). In total, 266 E. coli isolates were obtained from all the samples, with 67.3% being non-susceptible to at least one antibiotic tested and 6.7% multidrug resistant. The highest non-susceptibility was to ampicillin (48.1%) and the lowest non-susceptibility to ceftriaxone and azithromycin (0.8%). Significant non-susceptibility was observed to tetracycline (27.4%), nalidixic acid (20.3%), trimethoprim-sulfamethoxazole (13.9%), and chloramphenicol (11.7%) which have homologues used in the poultry industry. The most frequently observed resistance genes were bla CTX-M (100%), sul1 (80%), tetA (77%), and tetB (71%). ERIC-PCR grouped isolates into 27 clusters suggesting the spread of diverse clones across the farm-to-fork continuum. This reiterates the role of intensive poultry farming as a reservoir and a potential vehicle for the transmission of antibiotic resistance, with potentially severe public health implications, thus, requiring prompt and careful mitigation measures to protect human and environmental health.

Coagulase-negative staphylococci (CoNS) have engendered substantial interest in recent years as pathogenic causes of infections in both human and veterinary medicine, especially in the immunocompromised, critically ill, long-term hospitalized and in those harboring invasive medical devices such as catheters. They have been implicated in infections such as urinary tract infections, bloodstream infections, and invasive device-related infections, and are responsible for substantial economic losses in livestock production. The advancement of diagnostic techniques has increased our understanding of their molecular mechanisms of pathogenicity, even though distinguishing between innocuousness and pathogenicity is still challenging. The incidence of CoNS varied across the continent in humans and animals (mainly cattle), ranging from 6% to 68% in suspected human infections and from 3% to 61.7% in suspected animal infections, distributed across different geographic locations. Furthermore, there were varying antibiotic resistance patterns observed in CoNS isolates, with high methicillin resistance in some cases, leading to crossresistance against many antibiotics. Staphylococcus epidermidis , Staphylococcus haemolyticus , and Staphylococcus xylosus were most commonly reported in studies herein reviewed, while the enterotoxin C gene, atl E gene, ica gene, and hemolysin virulence factors were linked with enhanced pathogenicity. Advancement in identification and typing methods, including whole genome sequencing, virulence screening, and the assessment of the immune status of subjects in studies will help to thoroughly assess the true pathogenic potential of isolated CoNS species in developing countries. Careful antibiotic stewardship guidelines should be followed due to the ability of CoNS to develop multidrug resistance.

Aim: To investigate the dissemination of Salmonella spp. within four levels of government hospitals in KwaZulu-Natal, South Africa., Methods: The identification of Salmonella spp. was performed by amplification of the invA gene. Isolates were subjected to antimicrobial susceptibility testing and molecular characterization of eight resistance genes (qnrA, qnrB, qnrS, tetA, tetB, tetC, tetG, ermB) and three virulence genes (sitC, spvA, spv). Genetic relatedness between isolates was determined using enterobacterial repetitive intergenic consensus (ERIC) polymerase chain reaction., Findings: Ninety-four isolates were obtained. The largest source of isolates was the regional hospital. Paediatric wards had the highest prevalence of isolates. Nurses' tables contained the most isolates out of all sites sampled. Twenty-two clusters indicating diverse isolates were obtained via molecular typing. Four main ERIC types were identified, each unique to a specific hospital. A possibility of dissemination across the wards was noted as highly related isolates were present at various sites within the wards. Many of these sites were highly trafficked areas by healthcare staff. Ten multi-drug-resistant isolates were found., Conclusions: This study suggests that infection prevention and control strategies that involve environmental cleaning and decontamination may not be enough, or adhered to sufficiently, to prevent the dissemination of Salmonella spp., (Copyright © 2020 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.)

With the introduction of the One Health approach to global health advocated by the World Health Organization, the role of the environment as a reservoir and transmission route for diverse microorganisms is increasingly being recognised globally. This study investigated the diversity and functional profiles of bacterial communities using high-throughput metagenomics of the 16S rRNA gene in samples collected from environmental surfaces in different levels of healthcare in South Africa. A total of 150 samples were collected in three public hospitals [District (A), Regional (C) and Central (B)] from intensive care and paediatric wards. Military hospitals were excluded. Swabs were taken from mattresses, drip stands, ward telephones, patient files and sinks. A total of 7,996,346 reads were found, of which 7,319,569 were quality-filtered reads. Unique (and shared) microbial community structures were identified within the different hospital levels, locations and sample source. A total of 11 phyla, 29 classes, 50 orders, 105 families, 190 genera and 288 known species were identified. The primary phyla identified were Proteobacteria, Firmicutes and Actinobacteria. The dominant class identified was Gamma-proteobacteria, followed by Bacilli and Actinobacteria. Acinetobacter (16.08%), Citrobacter (13.64%), Staphylococcus (9.65%) and Corynebacterium (6.15%) were predominant genera. Although the functional profile analysis identified citrate cycle (TCA), signal transduction mechanisms, bisphenol degradation, tyrosine metabolism and transcription-factors as the dominant pathways, human disease functional classes, including involvement in antibiotic resistance, were significantly identified. The drip stands, patient files and ward telephones in all the wards of Hospitals A and C contained a higher number of human diseases functional classes. These findings highlight the potential of different hospital environments to serve as reservoirs and possible sources of bacterial pathogens; thus, the need for better monitoring and hygienic practices within the hospital environment., Competing Interests: Declaration of competing interest Professor Essack is Chairperson of the Global Respiratory Infection Partnership, sponsored by an unrestricted educational grant from Reckitt and Benckiser, UK. All the other authors declare no conflict of interest., (Copyright © 2020. Published by Elsevier B.V.)

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.

This study investigated the antibiotic resistance, virulence profiles, and clonality of Campylobacter jejuni and Campylobacter coli isolated from an intensive poultry farming system in KwaZulu-Natal, South Africa. Following ethical approval, samples were collected over six weeks using the farm-to-fork approach. Campylobacter spp. were identified using culture, confirmed and differentiated to species level by PCR, and subjected to antibiotic susceptibility testing. Selected antibiotic resistance (and mutations) and virulence genes were screened by PCR and confirmed by DNA sequencing. Genetic relatedness amongst the isolates was ascertained using pulsed-field gel electrophoresis. In all, 105 isolates were confirmed as belonging to both Campylobacter coli (60; 57%) and C. jejuni (45; 43%). The highest resistance was recorded against erythromycin and clindamycin. The gyrA mutation, A20175C/A2074G point mutation, tet(O), and cmeB, all associated with antibiotic resistance, were detected. All the virulence genes ( pldA , ciaB , cdtA, cdtB , cdtC , dnaJ , except for cadF ) were also detected. Isolates were grouped into five pulsotypes displaying 85% similarity, irrespective of their resistance profiles. The numerous permutations of clonality, antibiotic resistance, and virulence profiles evident in Campylobacter spp. pose a challenge to food safety and necessitate a comprehensive understanding of the molecular epidemiology of this organism to decrease its spread in the food chain., Competing Interests: S.Y.E. is the chairperson of the Global Respiratory Infection Partnership sponsored by an unrestricted educational grant from Reckitt and Benckiser (Pty.) Ltd. UK.

The poultry industry is among the main protein suppliers worldwide. Thus, this study determined the antibiotic resistance and virulence profiles of Enterococcus spp. along the farm-to-fork production chain of an intensive poultry system in the uMgungundlovu District, Kwazulu-Natal, South Africa. Overall, 162 samples along the continuum (growth phase, transport and post-slaughter) were evaluated for the presence of Enterococcus spp. using selective media, biochemical tests and polymerase chain reaction (PCR). Resistance profiles were assessed by Kirby-Bauer disk diffusion method following the WHO-AGISAR recommended antibiotics panel for Enterococcus spp. Antibiotic resistance and virulence genes were detected using real-time PCR. Clonal relatedness was evaluated by REP-PCR. Overall, 131 isolates were recovered across the continuum, (34% E. faecalis, 32% E. faecium, 2% E. gallinarum and 32% other Enterococcus spp.). Resistance to tetracycline (79%), erythromycin (70%), nitrofurantoin (18%), ampicillin (15%), streptomycin (15%), chloramphenicol (10%), ciprofloxacin (4%), tigecycline (4%), gentamicin (4%), teicoplanin (3%) was observed among all Enterococcus spp.; no vancomycin resistance (0%) was recorded. Also, 24% of E. faecium were resistant to quinupristin-dalfopristin. Twenty-four multidrug resistance (MDR) antibiograms were observed across all species; E. faecium (43%) showed the highest frequency of MDR. The most frequently observed antibiotic resistomes were tetM (76%) and ermB (66%) while smaller percentages were noted for aph(3')-IIIa (12%) and vanC1 (1%). Virulence genes efaAFs (100%), cpd (96%) and gelE (80%) were more prevalent in E. faecalis. Clonality revealed that isolates along the continuum were highly diverse with major REP-types consisting of isolates from the same sampling point. This study highlights the diversity of MDR Enterococcus in the food chain with isolates harbouring resistance and virulence genes. These could be reservoirs for the potential transfer of pathogenic enterococci carrying these genes from poultry to humans through the food chain continuum, thus, underscoring the need for routine antibiotic resistance surveillance in food animals., (Copyright © 2019 Elsevier B.V. All rights reserved.)

This study undertook genome mining and comparative genomics to gain genetic insights into the dominance of the methicillin-resistant Staphylococcus aureus (MRSA) endemic clone ST612-CC8-t1257-SCCmec&#95;IVd(2B), obtained from the poultry food chain in South Africa. Functional annotation of the genome revealed a vast array of similar central metabolic, cellular and biochemical networks within the endemic clone crucial for its survival in the microbial community. In-silico analysis of the clone revealed the possession of uniform defense systems, restriction-modification system (type I and IV), accessory gene regulator (type I), arginine catabolic mobile element (type II), and type 1 clustered, regularly interspaced, short palindromic repeat (CRISPR)Cas array (N = 7 ± 1), which offer protection against exogenous attacks. The estimated pathogenic potential predicted a higher probability (average P score ≈ 0.927) of the clone being pathogenic to its host. The clone carried a battery of putative virulence determinants whose expression are critical for establishing infection. However, there was a slight difference in their possession of adherence factors (biofilm operon system) and toxins (hemolysins and enterotoxins). Further analysis revealed a conserved environmental tolerance and persistence mechanisms related to stress (oxidative and osmotic), heat shock, sporulation, bacteriocins, and detoxification, which enable it to withstand lethal threats and contribute to its success in diverse ecological niches. Phylogenomic analysis with close sister lineages revealed that the clone was closely related to the MRSA isolate SHV713 from Australia. The results of this bioinformatic analysis provide valuable insights into the biology of this endemic clone.

Herein, we highlight the genome sequence of a novel Enterococcus faecalis sequence type 922 (ST922) strain isolated in South Africa. The 3,564,442-bp genome harbored defense systems, a resistome, a virulome, and genetic support, which is of importance to the control of hospital-acquired infections. The genomics of Enterococcus faecalis yields greater understanding into its pathogenesis., (Copyright © 2019 Shobo et al.)

Here, we describe the genomic sequence of a drug-resistant Staphylococcus aureus sequence type 239 (ST239) strain, SA9, isolated from a slaughterhouse chicken carcass in South Africa, including information about its antibiotic resistome, virulome, efflux genes, clonal lineage, and mobilome. This genomic information offers vital insights for the control of drug-resistant S. aureus ., (Copyright © 2019 Amoako et al.)

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.)

Extended spectrum β-lactamase-producing Enterobacteriaceae (ESBL-PE) represent a significant public health concern globally and are recognized by the World Health Organization as pathogens of critical priority. However, the prevalence of ESBL-PE in food animals and humans across the farm-to-plate continuum is yet to be elucidated in Sub-Saharan countries including Cameroon and South Africa. This work sought to determine the risk factors, carriage, antimicrobial resistance profiles and genetic relatedness of extended spectrum β-lactamase producing Enterobacteriaceae (ESBL-PE) amid pigs and abattoir workers in Cameroon and South Africa. ESBL-PE from pooled samples of 432 pigs and nasal and hand swabs of 82 humans were confirmed with VITEK 2 system. Genomic fingerprinting was performed by ERIC-PCR. Logistic regression (univariate and multivariate) analyses were carried out to identify risk factors for human ESBL-PE carriage using a questionnaire survey amongst abattoir workers. ESBL-PE prevalence in animal samples from Cameroon were higher than for South Africa and ESBL-PE carriage was observed in Cameroonian workers only. Nasal ESBL-PE colonization was statistically significantly associated with hand ESBL-PE (21.95% vs. 91.67%; p = 0.000; OR = 39.11; 95% CI 2.02⁻755.72; p = 0.015). Low level of education, lesser monthly income, previous hospitalization, recent antibiotic use, inadequate handwashing, lack of training and contact with poultry were the risk factors identified. The study highlights the threat posed by ESBL-PE in the food chain and recommends the implementation of effective strategies for antibiotic resistance containment in both countries.

The worldwide proliferation of life-threatening metallo-β-lactamase (MBL)-producing Gram-negative bacteria is a serious concern to public health. MBLs are compromising the therapeutic efficacies of β-lactams, particularly carbapenems, which are last-resort antibiotics indicated for various multidrug-resistant bacterial infections. Inhibition of enzymes mediating antibiotic resistance in bacteria is one of the major promising means for overcoming bacterial resistance. Compounds having potential MBL-inhibitory activity have been reported, but none are currently under clinical trials. The need for developing safe and efficient MBL inhibitors (MBLIs) is obvious, particularly with the continuous spread of MBLs worldwide. In this review, the emergence and escalation of MBLs in Gram-negative bacteria are discussed. The relationships between different class B β-lactamases identified up to 2017 are represented by a phylogenetic tree and summarized. In addition, approved and/or clinical-phase serine β-lactamase inhibitors are recapitulated to reflect the successful advances made in developing class A β-lactamase inhibitors. Reported MBLIs, their inhibitory properties, and their purported modes of inhibition are delineated. Insights into structural variations of MBLs and the challenges involved in developing potent MBLIs are also elucidated and discussed. Currently, natural products and MBL-resistant β-lactam analogues are the most promising agents that can become clinically efficient MBLIs. A deeper comprehension of the mechanisms of action and activity spectra of the various MBLs and their inhibitors will serve as a bedrock for further investigations that can result in clinically useful MBLIs to curb this global menace., (Copyright © 2018 American Society for Microbiology.)

Methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococcus (VRE) are included on the WHO high priority list of pathogens that require urgent intervention. Hence emphasis needs to be placed on developing novel class of molecules to tackle these pathogens. Teixobactin is a new class of antibiotic that has demonstrated antimicrobial activity against common bacteria. Here we examined the antimicrobial properties of three Teixobactin derivatives against clinically relevant bacterial isolates taken from South African patients. The minimum inhibitory concentration (MIC), the minimal bactericidal concentration (MBC), the effect of serum on MICs and the time-kill kinetics studies of our synthesized Teixobactin derivatives (3, 4, and 5) were ascertained following the CLSI 2017 guidelines and using the broth microdilution method. Haemolysis on red blood cells (RBCs) and cytotoxicity on peripheral blood mononuclear cells (PBMCs) were performed to determine the safety of these compounds. The MICs of 3, 4, and 5 against reference strains were 4-64 μg/ml, 2-64 μg/ml, and 0.5-64 μg/ml, respectively. The MICs observed for MRSA were (3) 32 μg/ml, (4) 2-4 μg/ml and (5) 2-4 μg/ml whilst those for VRE were (3) 8-16 μg/ml, (4) 4 μg/ml and (5) 2-16 μg/ml, respectively. In the presence of 50% human serum, there was no significant effect on the MICs. The compounds did not exhibit any effect on cell viability at their effective concentrations. Teixobactin derivatives (3, 4, and 5) inhibited bacterial growth in drug-resistant bacteria and hence emerge as potential antimicrobial agents. Molecular dynamic simulations suggested that the most dominant binding mode of Lys10-teixobactin (4) to lipid II is through the amide protons of the cycle, which is identical to data described in the literature for the natural teixobactin hence predicting the possibility of a similar mechanism of action.

The antileprosy drug clofazimine was recently repurposed as part of a newly endorsed short-course regimen for multidrug-resistant tuberculosis. It also enables significant treatment shortening when added to the first-line regimen for drug-susceptible tuberculosis in a mouse model. However, clofazimine causes dose- and duration-dependent skin discoloration in patients, and the optimal clofazimine dosing strategy in the context of the first-line regimen is unknown. We utilized a well-established mouse model to systematically address the impacts of duration, dose, and companion drugs on the treatment-shortening activity of clofazimine in the first-line regimen. In all studies, the primary outcome was relapse-free cure (culture-negative lungs) 6 months after stopping treatment, and the secondary outcome was bactericidal activity, i.e., the decline in the lung bacterial burden during treatment. Our findings indicate that clofazimine activity is most potent when coadministered with first-line drugs continuously throughout treatment and that equivalent treatment-shortening results are obtained with half the dose commonly used in mice. However, our studies also suggest that clofazimine at low exposures may have negative impacts on treatment outcomes, an effect that was evident only after the first 3 months of treatment. These data provide a sound evidence base to inform clofazimine dosing strategies to optimize the antituberculosis effect while minimizing skin discoloration. The results also underscore the importance of conducting long-term studies to allow the full evaluation of drugs administered in combination over long durations., (Copyright © 2018 American Society for Microbiology.)

Objectives: The anti-leprosy drug clofazimine has been shown to have antimicrobial activity against Mycobacterium tuberculosis and has been associated with treatment-shortening activity in both clinical and preclinical studies of TB chemotherapy. However, a reported lack of early bactericidal activity (EBA) in TB patients has raised questions regarding the usefulness of clofazimine as an anti-TB drug. Our objective was to systematically evaluate the EBA of clofazimine in vitro and in vivo to provide insight into how and when this drug exerts its antimicrobial activity against M. tuberculosis., Methods: We evaluated the 14 day EBA of clofazimine (i) in vitro at concentrations ranging from 4 times below to 4 times above the MIC for M. tuberculosis and (ii) in vivo in infected BALB/c mice at doses ranging from 1.5 to 100 mg/kg/day, and serum clofazimine levels were measured. In both experiments, isoniazid was used as the positive control., Results: In vitro, clofazimine, at any concentration tested, did not exhibit bactericidal activity during the first week of exposure; however, in the second week, it exhibited concentration-dependent antimicrobial activity. In vivo, clofazimine, at any dose administered, did not exhibit bactericidal activity during the first week, and limited antimicrobial activity was observed during the second week of administration. While serum clofazimine levels were clearly dose dependent, the antimicrobial activity was not significantly related to the dose administered., Conclusions: Our data suggest that clofazimine's delayed antimicrobial activity may be due more to its mechanism of action rather than to host-related factors., (© The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

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.)

A study was undertaken to determine the neuroprotective potential of Linezolid (LIN) in an animal model. Female Sprague-Dawley rats were either given a single (100 mg/kg) dose or treated daily for 4 weeks. A validated LC-MS/MS method was used to measure LIN levels in plasma and brain, this was paired with mass spectrometry imaging to determine the tissue spatial distribution of the drug. The results showed that after a single dose there was poor penetration of the drug into the brain. With multiple doses there were high tissue levels, with the drug reaching steady state in subsequent weeks. LIN displayed a promising distribution pattern with localisation in the brainstem. Systemic circulation is fed into the brain by the carotid and vertebral arteries which enter through the brain stem, therefore high drug concentrations is this area may protect against infectious agents entering via this route.

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.)

Experimental and clinical studies have indicated that the antileprosy drug clofazimine may contribute treatment-shortening activity when included in tuberculosis treatment regimens. Clofazimine accumulates to high levels in tissues, has a long half-life, and remains in the body for months after administration is stopped. We hypothesized that in tuberculosis treatment, accumulated clofazimine may contribute sustained antimicrobial activity after treatment cessation, and we used the BALB/c mouse model of chronic tuberculosis chemotherapy to address this hypothesis. Mycobacterium tuberculosis-infected mice were treated for 4 weeks or 8 weeks with either isoniazid alone, clofazimine alone, the first-line regimen rifampin-isoniazid-pyrazinamide-ethambutol, or a first-line regimen where clofazimine was administered in place of ethambutol. To evaluate posttreatment antimicrobial activity, bacterial regrowth in the lungs and spleens was assessed at the day of treatment cessation and 2, 4, 6, and 8 weeks after treatment was stopped. Bacterial regrowth was delayed in all mice receiving clofazimine, either alone or in combination, compared to the mice that did not receive clofazimine. This effect was especially evident in mice receiving multidrug therapy. In mice not receiving clofazimine, bacterial regrowth began almost immediately after treatment was stopped, while in mice receiving clofazimine, bacterial regrowth was delayed for up to 6 weeks, with the duration of sustained antimicrobial activity being positively associated with the time that serum clofazimine levels remained at or above the 0.25-μg/ml MIC for M. tuberculosis Thus, sustained activity of clofazimine may be important in the treatment-shortening effect associated with this drug., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Given the recent explosion of mass spectrometric imaging (MSI), it has become easier to assess drug tissue localisation without the use of radiolabeling and other more complex methods (such as PET and MRI). For MSI tissue preparation is of utmost importance, however, the lung in particular does pose some difficulties with imaging since it is made up of a number of air-filled alveoli. These organs are known to collapse when the thoracic cavity is pierced, losing its structural integrity and giving poor histological representation for drug distribution analysis. The use of cryoprotectants as a tissue inflation media will aid in the preservation of the lung's structural integrity during MSI experiments involving small molecule distribution. Various established cryoprotectants (DMSO, PvP, ethylene glycol, sucrose, DMEM, control serum, OCT) were selected as lung inflation media for MSI analysis of gatifloxacin (GAT). Female Sprague-Dawley rats were treated with GAT (10 mg/kg b.w) via i.p. injection. After 15 min the animals were terminated by halothane overdose, and each set of tissue inflated with a specific agent. Cryosections were made and MSI conducted to determine drug tissue distribution. During the early stages of the experimental procedure some crypreservatives were eliminated due to difficulties with sample preparation. While others displayed excellent preservation of the tissue structure and integrity. Following MSI analysis, some agents showed homogenous drug distribution while some displayed heterogeneous distribution favoring the basal periphery. Taking into account the physiology of the lung and previous MRI investigations of its perfusion, it is expected that a systemically administered drug would localize in the basal areas. DMSO and DMEM proved to display this distribution pattern while keeping structural integrity intact. However, the later was ruled out since it showed complete suppression of GAT in solution. From the cryoprotectants selected for this study, DMSO is the most promising lung inflation media focusing on small molecule distribution via MSI.

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.)

1. The penetration of tetracyclines into the brain has been widely documented. The aim of this work was to develop a matrix assisted laser desorption ionization-mass spectrometry imaging (MALDI MSI) method for the molecular histology of doxycycline (DOX) in the healthy rat brain. 2. The time-dependent distribution was investigated after an i.p. dose of 25 mg/kg at 0, 5, 30, 120, 240, 360 and 480 min postdose. LCMS/MS was used to quantify the drug in plasma and brain homogenates and MALDI MSI was used to determine the distribution of the analyte. 3. Within the first-hour postdose, the drug showed slow accumulation into the plasma and brain tissues. DOX brain concentration gradually increased and reached a peak (Cmax) of 1034.9 ng/mL at 240 min postdose, resulting in a brain plasma ratio of 31%. The images acquired by MSI matched the quantification results and clearly showed drug distribution over the entire rat brain coronal section from 5 min and its slow elimination after 360-min postdose. 4. Our findings confirm that MALDI MSI provides an advanced, label-free and faster alternative technique for xenobiotic distribution such as DOX in tissues, making it an essential drug discovery tool for other possible neuroprotective agents.

1. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) combines the sensitivity and selectivity of mass spectrometry with spatial analysis to provide a new dimension for histological analyses of the distribution of drugs in tissue. Pretomanid is a pro-drug belonging to a class of antibiotics known as nitroimidizoles, which have been proven to be active under hypoxic conditions and to the best of our knowledge there have been no studies investigating the distribution and localisation of this class of compounds in the brain using MALDI MSI. 2. Herein, we report on the distribution of pretomanid in the healthy rat brain after intraperitoneal administration (20 mg/kg) using MALDI MSI. Our findings showed that the drug localises in specific compartments of the rat brain viz. the corpus callosum, a dense network of neurons connecting left and right cerebral hemispheres. 3. This study proves that MALDI MSI technique has great potential for mapping the pretomanid distribution in uninfected tissue samples, without the need for molecular labelling.

This is the first report of clofazimine (CFZ) penetration and distribution in normal mouse brain. Mice were administered 25 mg/kg CFZ or 100 mg/kg CFZ orally, daily for 2 weeks. Animals were sacrificed and blood and brain tissues were harvested. Liquid chromatography tandem mass spectrometry showed high concentrations of CFZ in homogenized brain, with 100 mg/kg dose having significantly higher concentration than 25 mg/kg. Matrix-assisted laser desorption/ionization spectrometric imaging of brain sections showed widespread tissue distribution of CFZ. Our results show dose dependent localization in brain.

Rifampicin (RIF) is a major component for short-course chemotherapy against tuberculosis, since it is active against rapidly metabolizing as well as dormant bacteria. According to the Lipinski rules, RIF should not enter the blood-brain barrier. Visualization of tissue drug distribution is of major importance in pharmacological studies; thus, far imaging of RIF in the brain has been limited to positron emission tomography. We propose using matrix-assisted laser desorption/ionization mass spectrometry imaging techniques as a suitable alternative for the visualization and localization of drug tissue distribution. Using the liquid chromatography mass spectrometric (LCMS) technique, we were able to quantify the concentrations of RIF in the uninfected rat brain; we paired this with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) to show the time-dependent manner in which RIF is able to enter the brain. Our results show that even at the minute concentrations measured with LCMS/MS we were able visualize the drug and show its exact distribution in the rat brain. Other available methods require nuclear labeling and the detection of gamma rays produced by labeled compounds to visualize the compound and its localization; MALDI MSI is a more recently developed technique, which can provide detailed information on drug distribution in tissues when compared to other imaging techniques. This study shows that without any requirement for complex preprocessing we are able to produce images with a relatively improved resolution and localization than those acquired using more complex imaging methods, showing MALDI MSI to be an invaluable tool in drug distribution studies.