Membranome-based identification of amino acid substitution in Haemophilus influenzae multidrug efflux pump HmrM for reduced chloramphenicol susceptibility.

A decreased chloramphenicol susceptibility in Haemophilus influenzae is commonly caused by the activity of chloramphenicol acetyltransferases (CATs). However, the involvement of membrane proteins in chloramphenicol susceptibility in H. influenzae remains unclear. In this study, chloramphenicol susceptibility testing, whole-genome sequencing, and analyses of membrane-related genes were performed in 51 H. influenzae isolates. Functional complementation assays and structure-based protein analyses were conducted to assess the effect of proteins with sequence substitutions on the minimum inhibitory concentration (MIC) of chloramphenicol in CAT-negative H. influenzae isolates. Six isolates were resistant to chloramphenicol and positive for type A-2 CATs. Of these isolates, A3256 had a similar level of CAT activity but a higher chloramphenicol MIC relative to the other resistant isolates; it also had 163 specific variations in 58 membrane genes. Regarding the CAT-negative isolates, logistic regression and receiver operator characteristic curve analyses revealed that 48T > G (Asn16Lys), 85 C > T (Leu29Phe), and 88 C > A (Leu30Ile) in HI_0898 (emrA), and 86T > G (Phe29Cys) and 141T > A (Ser47Arg) in HI_1177 (artM) were associated with enhanced chloramphenicol susceptibility, whereas 997G > A (Val333Ile) in HI_1612 (hmrM) was associated with reduced chloramphenicol susceptibility. Furthermore, the chloramphenicol MIC was lower in the CAT-negative isolates with EmrA-Leu29Phe/Leu30Ile or ArtM-Ser47Arg substitution and higher in those with HmrM-Val333Ile substitution, relative to their counterparts. The Val333Ile substitution was associated with enhanced HmrM protein stability and flexibility and increased chloramphenicol MICs in CAT-negative H. influenzae isolates. In conclusion, the substitution in H. influenzae multidrug efflux pump HmrM associated with reduced chloramphenicol susceptibility was characterised.

SVEN_5003 is a Major Developmental Regulator in Streptomyces venezuelae.

Many regulatory genes that affect cellular development in Streptomyces, such as the canonical bld genes, have already been identified. However, in this study, we identified sven_5003 in Streptomyces venezuelae as a major new developmental regulatory gene, the deletion of which leads to a bald phenotype, typical of bld mutants, under multiple growth conditions. Our data indicated that disruption of sven_5003 also has a differential impact on the production of the two antibiotics jadomycin and chloramphenicol. Enhanced production of jadomycin but reduced production of chloramphenicol were detected in our sven_5003 mutant strain (S. venezuelae D5003). RNA-Seq analysis indicated that SVEN_5003 impacts expression of hundreds of genes, including genes involved in development, primary and secondary metabolism, and genes of unknown function, a finding confirmed by real-time PCR analysis. Transcriptional analysis indicated that sven_5003 is an auto-regulatory gene, repressing its own expression. Despite the evidence indicating that SVEN_5003 is a regulatory factor, a putative DNA-binding domain was not predicted from its primary amino acid sequence, implying an unknown regulatory mechanism by SVEN_5003. Our findings revealed that SVEN_5003 is a pleiotropic regulator with a critical role in morphological development in S. venezuelae.

Molecular basis of the persistence of chloramphenicol resistance among Escherichia coli and Salmonella spp. from pigs, pork and humans in Thailand.

This study aimed to investigate the potential mechanisms associated with the persistence of chloramphenicol (CHP) resistance in Escherichia coli and Salmonella enterica isolated from pigs, pork, and humans in Thailand. The CHP-resistant E. coli (n = 106) and Salmonella (n = 57) isolates were tested for their CHP susceptibility in the presence and absence of phenylalanine arginine ß-naphthylamide (PAßN). The potential co-selection of CHP resistance was investigated through conjugation experiments. Whole genome sequencing (WGS) was performed to analyze the E. coli (E329, E333, and E290) and Salmonella (SA448, SA461, and SA515) isolates with high CHP MIC (32-256 µg/mL) and predominant plasmid replicon types. The presence of PAßN significantly reduced the CHP MICs (≥4-fold) in most E. coli (67.9%) and Salmonella (64.9%). Ampicillin, tetracycline, and streptomycin co-selected for CHP-resistant Salmonella and E. coli-transconjugants carrying cmlA. IncF plasmids were mostly detected in cmlA carrying Salmonella (IncFIIAs) and E. coli (IncFIB and IncF) transconjugants. The WGS analysis revealed that class1 integrons with cmlA1 gene cassette flanked by IS26 and TnAs1 were located on IncX1 plasmid, IncFIA(HI1)/HI1B plasmids and IncFII/FIB plasmids. IncFIA(HI1)/HI1B/Q1in SA448 contained catA flanked by IS1B and TnAs3. In conclusion, cross resistance through proton motive force-dependent mechanisms and co-selection by other antimicrobial agents involved the persistence of CHP-resistance in E. coli in this collection. Dissemination of CHP-resistance genes was potentially facilitated by mobilization via mobile genetic elements.

Synthetic antimicrobial peptides: activity against vancomycin-resistant Enterococcus faecalis and modulation of chloramphenicol antibacterial activity.

AIMS: Enterococcus faecalis (E. faecalis) is a leading cause of nosocomial infection and presents a wide spectrum of antibiotic resistance, being vancomycin-resistant Enterococcus (VRE) one of the most relevant. Synthetic antimicrobial peptides (SAMPs) are currently a promising option to overcome antimicrobial resistance. Thus, the purpose of this study was to assess the effect of eight SAMPs against vancomycin-resistant E. faecalis, as well as to investigate their mechanism of action and synergy with conventional antibiotics. METHODS AND RESULTS: Here, eight SAMPs, Mo-CBP3-PepI, Mo-CBP3-PepII, Mo-CBP3-PepIII, RcAlb-PepI, RcAlb-PepII, RcAlb-PepIII, PepGAT, and PepKAA, were tested for antibacterial activity in vitro against E. faecalis (ATCC® 51299) through broth microdilution. A maximum of 48% of E. faecalis growth inhibition was achieved by treatment with SAMPs alone. However, when these peptides were combined with the antibiotic chloramphenicol, assessed by checkerboard method, the inhibition increased to 55%-76% of inhibition, two to three-folds of increase if compared to the effects of the compounds alone. Microscopic analysis showed that E. faecalis cells treated with a combination of SAMPs and chloramphenicol resulted in bacterial membrane damage. The biofilm inhibition maximum was 22% for SAMPs alone, when combined with chloramphenicol, the maximum increased to 33%. CONCLUSIONS: SAMPs and their combination with chloramphenicol demonstrate antibacterial activity against E. faecalis, possibly by inducing bacterial membrane damage.

Nanoscale zero-valent iron reverses resistance of Pseudomonas aeruginosa to chloramphenicol.

Zero-valent iron (ZVI) has been extensively studied for its capacity to remove various contaminants in the environments. However, whether ZVI affects bacterial resistance to antibiotics has not been fully explored. Herein, it was unexpected that, compared with microscale ZVI (mZVI), nanoscale ZVI (nZVI) facilitated the susceptibility of Pseudomonas aeruginosa (P. aeruginosa) to chloramphenicol (CAP), with a decrease in the minimal inhibitory concentration (MIC) of about 60 %, demonstrating a nanosize-specific effect. nZVI enhanced CAP accumulation in P. aeruginosa via inhibitory effect on efflux pumps activated by MexT, thus conferring the susceptibility of P. aeruginosa to CAP. Circular dichroism spectroscopy revealed that the structure of MexT was changed during the evolution. More importantly, molecular dynamic simulations uncovered that, once the structure of MexT changed, it would be more likely to interact with nZVI, resulting in more serious changes in its secondary structure, which was consistent with the increasing susceptibility of P. aeruginosa to CAP. Collectively, this study elucidated the size-specific effect and the underlying mechanism of ZVI on the bacterial evolution of susceptibility toward antibiotics, highlighting the potentials of nZVI-based technologies on the prevention of bacterial resistance to antibiotics, one of the most important issue for globally public health.

Design, optimization and pharmaceutical characterization of wound healing film dressings with chloramphenicol and ibuprofen.

OBJECTIVE: The aim of the present study was to develop and optimize a wound dressing film loaded with chloramphenicol (CAM) and ibuprofen (IBU) using a Quality by Design (QbD) approach. SIGNIFICANCE: The two drugs have been combined in the same dressing as they address two critical aspects of the wound healing process, namely prevention of bacterial infection and reduction of inflammation and pain related to injury. METHODS: Three critical formulation variables were identified, namely the ratios of Kollicoat SR 30D, polyethylene glycol 400 and polyvinyl alcohol. These variables were further considered as factors of an experimental design, and 17 formulations loaded with CAM and IBU were prepared via solvent casting. The films were characterized in terms of dimensions, mechanical properties and bioadhesion. Additionally, the optimal formulation was characterized regarding tensile properties, swelling behavior, water vapor transmission rate, surface morphology, thermal behavior, goniometry, in vitro drug release, cell viability, and antibacterial activity. RESULTS: The film was optimized by setting minimal values for the folding endurance, adhesive force and hardness. The optimally formulated film showed good fluid handling properties in terms of swelling behavior and water vapor transmission rate. IBU and CAM were released from the film up to 80.9% and 82.5% for 8 h. The film was nontoxic, and the antibacterial activity was prominent against Micrococcus spp. and Streptococcus pyogenes. CONCLUSIONS: The QbD approach was successfully implemented to develop and optimize a novel film dressing promising for the treatment of low-exuding acute wounds prone to infection and inflammation.

Bacitracin-resistant Staphylococcus aureus induced in chicken gut and in vitro under bacitracin exposure.

It is common knowledge that prolonged and excessive use of antibiotics can lead to antimicrobial resistance. However, the characteristics and mechanism of resistant-bacteria induced by clinically recommended and prophylactic dose drugs remain largely unclear. This study aimed to observe the trends of drug resistance of the bacitracin-susceptible Staphylococcus aureus strain FS127 under exposure to bacitracin (BAC), which were induced in vitro and in chicken gut. Antimicrobial susceptibility testing was used to detect the susceptibility of S. aureus induced in vitro and in the chicken gut to gentamicin, chloramphenicol, tetracycline, doxycycline, penicillin and chloramphenicol. The research results showed that bacitracin could induce drug resistance in S. aureus both in vitro and in vivo. The bacitracin-resistance rate of S. aureus isolated from chicken gut was positively correlated with the dose and time of bacitracin administration. The findings revealed that bacitracin-resistant S. aureus induced in vivo had enhanced susceptibility to chloramphenicol but no such change in vitro. Meanwhile, RT-qPCR assay was used to detect the expression levels of vraD, braD, braR and bacA in typical strains with different bacitracin-resistance levels. It was found that BacA may play a key role in the bacitracin resistance of S. aureus. In conclusion, this work reveals the characteristics and mechanism of bacitracin-resistant S. aureus induced by bacitracin in vivo and in vitro respectively.

Chloramphenicol Binding Sites of Acinetobacter baumannii Chloramphenicol Acetyltransferase CatB8.

Acinetobacter baumannii is a multidrug-resistant pathogen that has become one of the most challenging pathogens in global healthcare. Several antibiotic-resistant genes, including catB8, have been identified in the A. baumannii genome. CatB8 protein, one of the chloramphenicol acetyltransferases (Cats), is encoded by the catB8 gene. Cats can convert chloramphenicol (chl) to 3-acetyl-chl, leading to bacterial resistance to chl. Here, we present the high-resolution cocrystal structure of CatB8 with chl. The structure that we resolved showed that each monomer of CatB8 binds to four chl molecules, while its homologous protein only binds to one chl molecule. One of the newly discovered chl binding site overlaps with the site of another substrate, acetyl-CoA. Through structure-based biochemical analyses, we identified key residues for chl recruiting and acetylation of chl in CatB8. Our work is of significant importance for understanding the drug resistance of A. baumannii and the effectiveness of antibiotic treatment.

Evaluation of the in vitro effects of concentrations of antibiotics on three Enterobacteriaceae isolates.

Due to the misuse and overuse of antibiotics, bacteria are now exposed to sub-minimum inhibitory concentrations (sub-MICs) of antibiotics in various environments. In recent years, exposure of bacteria to sub-MICs of antibiotics has led to the widespread emergence of antibiotic-resistant bacteria. In this study, three bacterial species from the Enterobacteriaceae family (Raoultella ornithinolytica, Pantoea agglomerans and Klebsiella quasivariicola) were isolated from water. The antibiotic susceptibility of these bacteria to 16 antibiotics was then investigated. The effects of sub-MICs of four selected antibiotics (kanamycin, chloramphenicol, meropenem, and ciprofloxacin) on the growth, biofilm formation, surface polysaccharide production, siderophore production, morphology, and expression of the translational/transcriptional regulatory transformer gene rfaH of these bacteria were analysed. The MICs of kanamycin, chloramphenicol, meropenem, and ciprofloxacin were determined to be 1, 2, 0.03 and 0.03 µg/mL for R. ornithinolytica; 0.6, 6, 0.03 and 0.05 µg/mL for P. agglomerans; and 2, 5, 0.04 and 0.2 µg/mL for K. quasivariicola. The growth kinetics and biofilm formation ability decreased for all three isolates at sub-MICs. The surface polysaccharides of R. ornithinolytica and P. agglomerans increased at sub-MICs. There was no significant change in the siderophore activities of the bacterial isolates, with the exception of MIC/2 meropenem in R. ornithinolytica and MIC/2 kanamycin in K. quasivariicola. It was observed that the sub-MICs of meropenem and ciprofloxacin caused significant changes in bacterial morphology. In addition, the expression of rfaH in R. ornithinolytica and K. quasivariicola increased with the sub-MICs of the selected antibiotics.

Quantifying Dynamic Phenotypic Heterogeneity in Resistant Escherichia coli under Translation-Inhibiting Antibiotics.

Understanding the phenotypic heterogeneity of antibiotic-resistant bacteria following treatment and the transitions between different phenotypes is crucial for developing effective infection control strategies. The study expands upon previous work by explicating chloramphenicol-induced phenotypic heterogeneities in growth rate, gene expression, and morphology of resistant Escherichia coli using time-lapse microscopy. Correlating the bacterial growth rate and cspC expression, four interchangeable phenotypic subpopulations across varying antibiotic concentrations are identified, surpassing the previously described growth rate bistability. Notably, bacterial cells exhibiting either fast or slow growth rates can concurrently harbor subpopulations characterized by high and low gene expression levels, respectively. To elucidate the mechanisms behind this enhanced heterogeneity, a concise gene expression network model is proposed and the biological significance of the four phenotypes is further explored. Additionally, by employing Hidden Markov Model fitting and integrating the non-equilibrium landscape and flux theory, the real-time data encompassing diverse bacterial traits are analyzed. This approach reveals dynamic changes and switching kinetics in different cell fates, facilitating the quantification of observable behaviors and the non-equilibrium dynamics and thermodynamics at play. The results highlight the multi-dimensional heterogeneous behaviors of antibiotic-resistant bacteria under antibiotic stress, providing new insights into the compromised antibiotic efficacy, microbial response, and associated evolution processes.

Effect of Chloramphenicol as Antibiotic on the Structure and Function of Pepsin and Its Mechanism of Action.

The interaction between chloramphenicol (CHL) and pepsin (PEP), as well as the impact of CHL on PEP conformation, were investigated using spectroscopic techniques and molecular docking simulations in this study. The experimental results demonstrate that CHL exhibits a static quenching effect on PEP. The thermodynamic parameters indicate that the reaction between CHL and PEP is spontaneous, primarily driven by hydrogen bonding and van der Waals forces. Moreover, the binding distance of r<7 nm suggests the occurrence of Förster's non-radiative energy transfer between these two molecules. In the synchronous fluorescence spectrum, the maximum fluorescence intensity of PEP produced a redshift phenomenon, indicating that CHL was bound to tryptophan residues of PEP. The addition of CHL induces changes in the secondary structure of PEP, as confirmed by the observed alterations in peak values in three-dimensional fluorescence spectra. The UV spectra reveal a redshift of 3 nm in the maximum absorption peak, indicating a conformational change in the secondary structure of PEP upon addition of CHL. Circular dichroism analysis demonstrates significant alterations in the α-helix, ß-sheet, ß-turn, and random coil contents of PEP before and after CHL incorporation, further confirming its ability to modulate the secondary structure of PEP.

A review on the antibiotic florfenicol: Occurrence, environmental fate, effects, and health risks.

Florfenicol, as a replacement for chloramphenicol, can tightly bind to the A site of the 23S rRNA in the 50S subunit of the 70S ribosome, thereby inhibiting protein synthesis and bacterial proliferation. Due to the widespread use in aquaculture and veterinary medicine, florfenicol has been detected in the aquatic environment worldwide. Concerns over the effects and health risks of florfenicol on target and non-target organisms have been raised in recent years. Although the ecotoxicity of florfenicol has been widely reported in different species, no attempt has been made to review the current research progress of florfenicol toxicity, hormesis, and its health risks posed to biota. In this study, a comprehensive literature review was conducted to summarize the effects of florfenicol on various organisms including bacteria, algae, invertebrates, fishes, birds, and mammals. The generation of antibiotic resistant bacteria and spread antibiotic resistant genes, closely associated with hormesis, are pressing environmental health issues stemming from overuse or misuse of antibiotics including florfenicol. Exposure to florfenicol at µg/L-mg/L induced hormetic effects in several algal species, and chromoplasts might serve as a target for florfenicol-induced effects; however, the underlying molecular mechanisms are completely lacking. Exposure to high levels (mg/L) of florfenicol modified the xenobiotic metabolism, antioxidant systems, and energy metabolism, resulting in hepatotoxicity, renal toxicity, immunotoxicity, developmental toxicity, reproductive toxicity, obesogenic effects, and hormesis in different animal species. Mitochondria and the associated energy metabolism are suggested to be the primary targets for florfenicol toxicity in animals, albeit further in-depth investigations are warranted for revealing the long-term effects (e.g., whole-life-cycle impacts, multigenerational effects) of florfenicol, especially at environmental levels, and the underlying mechanisms. This will facilitate the evaluation of potential hormetic effects and construction of adverse outcome pathways for environmental risk assessment and regulation of florfenicol.

Development of membrane-targeting TPP+-chloramphenicol conjugates to combat methicillin-resistant staphylococcus aureus (MRSA) infections.

Infections caused by drug-resistant bacteria have become a new challenge in infection treatment, gravely endangering public health. Chloramphenicol (CL) is a well-known antibiotic which has lost its efficacy due to bacterial resistance. To address this issue, herein we report the design, synthesis and biological evaluations of novel triphenylphosphonium chloramphenicol conjugates (TPP+-CL). Study results indicated that compounds 39 and 42 possessed remarkable antibacterial effects against clinically isolated methicillin-resistant Staphylococcus aureus (MRSA) with MIC values ranging from 1 to 2 µg/mL, while CL was inactive to the tested MRSA strains. In addition, these conjugates exhibited rapid bactericidal properties and low toxicity, and did not readily induced bacterial resistance, obviously outperforming the parent drug CL. In a mouse model infected with a clinically isolated MRSA strain, compound 39 at a dose of 20 mg/kg exhibited a comparable or even better in vivo anti-MRSA efficacy than the golden standard drug vancomycin, while no toxicity was observed.

Synergistic antibacterial effects of exopolysaccharides/nickel-nanoparticles composites against multidrug-resistant bacteria.

The need for an alternative treatment to fight infectious diseases caused by antibiotic-resistant bacteria is increasing. A possible way to overcome bacterial resistance to antibiotics is by reintroducing commonly used antibiotics with a sensitizer capable of enhancing their antimicrobial effect in resistant bacteria. Here, we use a composite composed of exopolysaccharide capped-NiO NPs, with antimicrobial effects against antibiotic-resistant Gram-positive and Gram-negative bacteria. It potentiated the antimicrobial effects of four different antibiotics (ampicillin, kanamycin, chloramphenicol, and ciprofloxacin) at lower concentrations than their minimal inhibitory concentrations. We observed that the Ni-composite synergistically enhanced, fourfold, the antibacterial effect of kanamycin and chloramphenicol against multidrug-resistant Staphylococcus aureus and Pseudomonas aeruginosa, as well as ampicillin against multidrug-resistant Staphylococcus aureus, and ciprofloxacin against multidrug-resistant Pseudomonas aeruginosa by eightfold. We also found that Ni-composite could not inhibit biofilm synthesis on the tested bacterial strains. Our results demonstrated the possibility of using metal nanoparticles, like NiO, as a sensitizer to overcome bacterial antibiotic resistance.

Synergistic effect of two antimicrobial peptides, BP203 and MAP-0403 J-2 with conventional antibiotics against colistin-resistant Escherichia coli and Klebsiella pneumoniae clinical isolates.

Drug-resistant Enterobacterales infections are a great health concern due to the lack of effective treatments. Consequently, finding novel antimicrobials or combining therapies becomes a crucial approach in addressing this problem. BP203 and MAP-0403 J-2, novel antimicrobial peptides, have exhibited effectiveness against Gram-negative bacteria. In this study, we assessed the in vitro antibacterial activity of BP203 and MAP-0403 J-2, along with their synergistic interaction with conventional antibiotics including colistin, rifampicin, chloramphenicol, ceftazidime, meropenem, and ciprofloxacin against colistin-resistant Escherichia coli and Klebsiella pneumoniae clinical isolates. The minimal inhibitory concentrations (MIC) of BP203 and MAP-0403 J-2 against tested E. coli isolates were 2-16 and 8-32 µg/mL, respectively. However, for the majority of K. pneumoniae isolates, the MIC of BP203 and MAP-0403 J-2 were >128 µg/mL. Notably, our results demonstrated a synergistic effect when combining BP203 with rifampicin, meropenem, or chloramphenicol, primarily observed in most K. pneumoniae isolates. In contrast, no synergism was evident between BP203 and colistin, chloramphenicol, ceftazidime, rifampicin, or ciprofloxacin when tested against all E. coli isolates. Furthermore, synergistic effects between MAP-0403 J-2 and rifampicin, ceftazidime or colistin were observed against the majority of E. coli isolates. Similarly, the combined effect of MAP-0403 J-2 with rifampicin or chloramphenicol was synergistic in the majority of K. pneumoniae isolates. Importantly, these peptides displayed the stability at high temperatures, across a wide range of pH values, in specific serum concentrations and under physiological salt conditions. Both peptides also showed no significant hemolysis and cytotoxicity against mammalian cells. Our findings suggested that BP203 and MAP-0403 J-2 are promising candidates against colistin-resistant E. coli. Meanwhile, the synergism of these peptides and certain antibiotics could be of great therapeutic value as antimicrobial drugs against infections caused by colistin-resistant E. coli and K. pneumoniae.

Blue Light Potentiates Antibiotics in Bacteria via Parallel Pathways of Hydroxyl Radical Production and Enhanced Antibiotic Uptake.

In the age of antimicrobial resistance, the urgency by which novel therapeutic approaches need to be introduced into the clinical pipeline has reached critical levels. Antimicrobial blue light (aBL), as an alternative approach, has demonstrated promise as a stand-alone therapeutic method, albeit with a limited window of antimicrobial activity. Work by others indicates that treatment with antibiotics increases the production of reactive oxygen species (ROS) which may, in part, contribute to the bactericidal effects of antibiotics. These findings suggest that there may be potential for synergistic interactions with aBL, that similarly generates ROS. Therefore, in this study, the mechanism of aBL is investigated, and the potential for aBL to synergistically promote antibiotic activity is similarly evaluated. Furthermore, the translatability of using aBL and chloramphenicol in combination within a mouse model of Acinetobacter baumanii burn infection is assessed. It is concluded that porphyrins and hydroxyl radicals driven by "free iron" are paramount to the effectiveness of aBL; and aBL is effective at promoting multiple antibiotics in different multidrug-resistant bacteria. Moreover, rROS up-regulation, and promoted antibiotic uptake are observed during aBL+antibiotic exposure. Lastly, aBL combined with chloramphenicol appears to be both effective and safe for the treatment of A. baumannii burn infection. In conclusion, aBL may be a useful adjunct therapy to antibiotics to potentiate their action.

Antimicrobial resistance profiles of salmonella spp. and escherichia coli isolated from fresh nile tilapia (oreochromis niloticus) fish marketed for human consumption.

BACKGROUND: Salmonella spp. and pathogenic strains of Escherichia coli are among the major foodborne zoonotic pathogens. These bacterial pathogens cause human illnesses characterized by hemorrhagic colitis, vomiting, nausea, and other agent-related symptoms. The increasing occurrence of antimicrobial resistance in these pathogens is also a serious public health concern globally. Regular surveillance of phenotypes and genotypes of Salmonella spp. and Escherichia coli from animal-derived foods is necessary for effective reduction and control of these foodborne pathogens. This study was conducted to assess the occurrence, antimicrobial resistance, virulence genes and genetic diversity of Salmonella spp. and E. coli isolates from fresh Nile tilapia obtained from retail markets in Nairobi, Kenya. METHODS: A total of 68 fresh Nile tilapia fish samples were collected from retail markets and used for isolation of Salmonella spp. and E. coli. Antimicrobial susceptibilities of the isolates weretested by Kirby-Bauer agar disc diffusion method. According to the antimicrobial resistance profiles, the multi-drug resistant isolates were identified by 16 S rRNA sequencing and phylogenetic analysis using the Bayesian inference method. The MDR Salmonella spp. and E. coli isolates were subjected to PCR-based screening for the detection virulence and antibiotic resistance genes. RESULTS: The prevalence of contamination of the fish samples with Salmonella spp. and E.coli was 26.47% and 35.29% respectively. Overall phenotypic resistance among the Salmonella spp. ranged from 5.5% for ceftazidime, chloramphenicol, meropenem, nitrofurantoin and streptomycin and 22.2% for penicillin-G. For E. coli phenotypic resistance ranged from 4.2% for ceftazidime and chloramphenicol and 25% for rifampicin. Multi-drug resistance was observed in three Salmonella spp. and two E. coli isolates. Results of 16 S rRNA sequences, sequence alignment and phylogenic trees confirmed the identified MDR isolates as S. typhymurium WES-09, S. typhymurium MAK-22, S. typhimurium EMB-32 and E. coli MAK-26 and E. coli LAN-35. The presence of antibiotic-resistance genes belonging to ß-lactamases, tetracycline, sulfonamide, trimethoprim and aminoglycosides-resistant genes were detected in all the identified MDR isolates. CONCLUSIONS: The findings from this study indicate that Nile tilapia (Oreochromis niloticus) sold in retail markets can acts as reservoirs of Salmonella spp. and E. coli pathogens linked to human disease, some of which were multidrug resistance to critically important antimicrobials. Both microorganisms are of zoonotic significance and represent a significant public health risk to the society.

Unveiling synergism of polymyxin B with chloramphenicol derivatives against multidrug-resistant (MDR) Klebsiella pneumoniae.

Polymyxins are last-line antibiotics against multidrug-resistant Klebsiella pneumoniae but using polymyxins alone may not be effective due to emerging resistance. A previous study found that combining polymyxin B with chloramphenicol effectively kills MDR K. pneumoniae, although the bone marrow toxicity of chloramphenicol is concerning. The aim of this study is to assess the antibacterial efficacy and cytotoxicity of polymyxin B when combined with chloramphenicol and its derivatives, namely thiamphenicol and florfenicol (reported to have lesser toxicity compared to chloramphenicol). The antibacterial activity was evaluated with antimicrobial susceptibility testing using broth microdilution and time-kill assays, while the cytotoxic effect on normal bone marrow cell line, HS-5 was evaluated using the MTT assay. All bacterial isolates tested were found to be susceptible to polymyxin B, but resistant to chloramphenicol, thiamphenicol, and florfenicol when used alone. The use of polymyxin B alone showed bacterial regrowth for all isolates at 24 h. The combination of polymyxin B and florfenicol demonstrated additive and synergistic effects against all isolates (≥ 2 log10 cfu ml-1 reduction) at 4 and 24 h, respectively, while the combination of polymyxin B and thiamphenicol resulted in synergistic killing at 24 h against ATCC BAA-2146. Furthermore, the combination of polymyxin B with florfenicol had the lowest cytotoxic effect on the HS-5 cells compared to polymyxin B combination with chloramphenicol and thiamphenicol. Overall, the combination of polymyxin B with florfenicol enhanced bacterial killing against MDR K. pneumoniae and exerted minimal cytotoxic effect on HS-5 cell line.

Combined antimicrobial effect of phage-derived endolysin and depolymerase against biofilm-forming Salmonella Typhimurium.

This study was designed to evaluate the antimicrobial activity of phage-derived endolysin (LysPB32) and depolymerase (DpolP22) against planktonic and biofilm cells of Salmonella Typhimurium (STKCCM). Compared to the control, the numbers of STKCCM were reduced by 4.3 and 5.9 log, respectively, at LysPB32 and LysPB32 + DpolP22 in the presence of polymyxin B (PMB) after 48-h incubation at 37 °C. LysPB32 + DpolP22 decreased the relative fitness (0.8) and the cross-resistance of STKCCM to chloramphenicol (CHL), cephalothin (CEP), ciprofloxacin (CIP), and tetracycline (TET) in the presence of PMB. The MICtrt/MICcon ratios of CHL, CEP, CIP, PMB, and TET were between 0.25 and 0.50 for LysPB32 + DpolP22 in the presence of PMB. These results suggest that the application of phage-encoded enzymes with antibiotics can be a promising approach for controlling biofilm formation on medical and food-processing equipment. This is noteworthy in that the application of LysPB32 + DpolP22 could increase antibiotic susceptibility and decrease cross-resistance to other antibiotics.

Development of pBACpAK entrapment vector derivatives to detect intracellular transfer of mobile genetic elements within chloramphenicol resistant bacterial isolates.

Antimicrobial resistance disseminates throughout bacterial populations via horizontal gene transfer, driven mainly by mobile genetic elements (MGEs). Entrapment vectors are key tools in determining MGE movement within a bacterial cell between different replicons or between sites within the same replicon. The pBACpAK entrapment vector has been previously used to study intracellular transfer in Gram-negative bacteria however since pBACpAK contains a chloramphenicol resistance gene, it cannot be used in bacterial isolates which are already resistant to chloramphenicol. Therefore, we developed new derivatives of the pBACpAK entrapment vector to determine intracellular transfer of MGEs in an Escherichia coli DH5α transconjugant containing the chloramphenicol resistance plasmid pD25466. The catA1 of pBACpAK was replaced by both mcr-1 in pBACpAK-COL and aph(3')-Ia in pBACpAK-KAN, allowing it to be used in chloramphenicol resistant strains. The plasmid constructs were verified and then used to transform the E. coli DH5α/pD25466 transconjugants in order to detect intracellular movement of the MGEs associated with the pD25466 plasmid. Here we report on the validation of the expanded suite of pBACpAK vectors which can be used to study the intracellular transfer of MGEs between, and within, replicons in bacteria with different antimicrobial resistance profiles.