Leveraging the potential of bacterial lateral gene transfer in boosting the efficacy of an edible probiotic prototype yogurt vaccine for COVID-19.

Administration of coronavirus disease-2019 (COVID-19) vaccines with appropriate booster doses through painful injections under clinical supervision was challenging during the recent COVID-19 pandemic. As an alternative solution, we designed a safer, edible probiotic yogurt vaccine prototype (YoVac) that can be orally consumed by circumventing painful injections and clinical supervision. We hypothesized that YoVac prepared using Lactobacillus carrying an antigen coding gene (donor) can transfer the same to other bacteria (recipients) in the human gut microbiome (hgMb) through lateral gene transfer (LGT) for boosted antigen levels potentially triggering a robust immune response. In this study we confirmed the in vitro LGT efficiency of a plasmid (pRBD-Ampr) containing severe acute respiratory syndrome coronavirus-2 (SARS CoV-2) spike protein-receptor binding domain (RBD) coding gene along with an ampicillin-resistance gene (selection marker) from the probiotic Lactobacillus (donor) cultured from homemade yogurt to E. coli and Helicobacter pylori (recipients). Both the donor and recipient bacteria not only exhibited ampicillin-resistance from pRBD-Ampr but also expressed RBD protein. Furthermore, Lactobacillus isolated from YoVac consistently showed the expression of RBD protein over a period of one month confirming the shelf life of our prototype stored at 4 °C. Taken together, our in vitro results provide a preliminary basis for the potential in vivo transfer of RBD coding gene from YoVac to other bacterial species in the hgMb through LGT and may potentially boost the vaccine dosage by delegating them.

Linezolid- and Multidrug-Resistant Enterococci in Raw Commercial Dog Food, Europe, 2019-2020.

We describe enterococci in raw-frozen dog food commercialized in Europe as a source of genes encoding resistance to the antibiotic drug linezolid and of strains and plasmids enriched in antibiotic-resistance and virulence genes in hospitalized patients. Whole-genome sequencing was fundamental to linking isolates from dog food to human cases across Europe.

LC-MS/MS-based metabolic profiling of Escherichia coli under heterologous gene expression stress.

Escherichia coli is frequently exploited for genetic manipulations and heterologous gene expression studies. We have evaluated the metabolic profile of E. coli strain BL21 (DE3) RIL CodonPlus after genetic modifications and subjecting to the production of recombinant protein. Three genetically variable E. coli cell types were studied, normal cells (susceptible to antibiotics) cultured in simple LB medium, cells harboring ampicillin-resistant plasmid pET21a (+), grown under antibiotic stress, and cells having recombinant plasmid pET21a (+) ligated with bacterial lactate dehydrogenase gene grown under ampicillin and standard isopropyl thiogalactoside (IPTG)-induced gene expression conditions. A total of 592 metabolites were identified through liquid chromatography-mass spectrometry/mass spectrometry analysis, feature and peak detection using XCMS and CAMERA followed by precursor identification by METLIN-based procedures. Overall, 107 metabolites were found differentially regulated among genetically modified cells. Quantitative analysis has shown a significant modulation in DHNA-CoA, p-aminobenzoic acid, and citrulline levels, indicating an alteration in vitamin K, folic acid biosynthesis, and urea cycle of E. coli cells during heterologous gene expression. Modulations in energy metabolites including NADH, AMP, ADP, ATP, carbohydrate, terpenoids, fatty acid metabolites, diadenosine tetraphosphate (Ap4A), and l-carnitine advocate major metabolic rearrangements. Our study provides a broader insight into the metabolic adaptations of bacterial cells during gene manipulation experiments that can be prolonged to improve the yield of heterologous gene products and concomitant production of valuable biomolecules.

Epidemiology of Haemophilus influenzae in the Republic of Ireland, 2010-2018.

The purpose of this study was to investigate H. influenzae epidemiology in the Republic of Ireland. We performed serotyping, multi-locus sequence typing (MLST) and susceptibility testing on H. influenzae isolates received by the Irish Meningitis and Sepsis Reference Laboratory from 2010 to 2018. Three hundred sixty-seven invasive and 41 non-invasive infection (NII) isolates were received. Invasive isolates were mostly recovered from paediatric (21%) and elderly (42%) populations. Invasive disease was more prevalent in females of childbearing age (72%) compared with males the same age (28%). Non-typeable H. influenzae (NTHi) predominated among invasive (83%) and NII (95%). Invasive Hib disease isolates were infrequent (4%, n = 15). Among invasive disease, Hif was the commonest encapsulated serotype (10%, n = 37), and the only encapsulated serotype detected in NII (5%, 2/41). The first PCR-confirmed serotypes d and a in Ireland were characterised among invasive disease in 2017 and 2018, respectively. MLST revealed a diverse NTHi population, while encapsulated serotypes were clonal. Sequence type (ST) 103 (n = 14) occurred exclusively in invasive NTHi disease. Ampicillin resistance (AmpR) was 18% among invasive isolates and 22% in NII. ß-Lactamase production was the main source of ampicillin resistance in invasive and NII isolates. We detected ß-lactamase negative ampicillin resistance (BLNAR) among invasive isolates. We report an NTHi fluoroquinolone-resistant clone: ST1524 among invasive (n = 2) and NII isolates (n = 2). The Hib vaccine has positively impacted on Hib disease in Ireland, given the low frequency of Hib. The dominance of NTHi, emergence of serotypes a and d and BLNAR suggest a changing H. influenzae epidemiology in Ireland.

Invasive non-typeable Haemophilus influenzae infection due to endometritis associated with adenomyosis.

BACKGROUND: The widespread administration of the Haemophilus influenzae type b vaccine has led to the predominance of non-typable H. influenzae (NTHi). However, the occurrence of invasive NTHi infection based on gynecologic diseases is still rare. CASE PRESENTATION: A 51-year-old Japanese woman with a history of adenomyoma presented with fever. Blood cultures and a vaginal discharge culture were positive with NTHi. With the high uptake in the uterus with 67Ga scintigraphy, she was diagnosed with invasive NTHi infection. In addition to antibiotic administrations, a total hysterectomy was performed. The pathological analysis found microabscess formations in adenomyosis. CONCLUSIONS: Although NTHi bacteremia consequent to a microabscess in adenomyosis is rare, this case emphasizes the need to consider the uterus as a potential source of infection in patients with underlying gynecological diseases, including an invasive NTHi infection with no known primary focus.

The non-coding RNA rprA can increase the resistance to ampicillin in Escherichia coli.

OBJECTIVES: The non-coding RNA rprA can increase the resistance to ampicillin in Escherichia coli. METHODS: Bacterial DNA was extracted by boiling method and then amplified using polymerase chain reaction (PCR) with two different primer sets. Recombinant pET28a/rprA-sense and -antisense plasmids were separately transferred into the competent E. coli BL21 (DE3) by chemical methods using heat shock. The expression was analyzed at the RNA level using Semi quantitative RT PCR. The turbidity difference between the bacteria was checked by Broth Dilution method. RESULTS: The statistical analysis showed that the turbidity difference between the up regulated and control bacteria is significant (p value < 0.0001). The ANOVA test also showed the significant difference between the down regulated and control bacteria (p value < 0.0001). CONCLUSION: Considering this mechanism, there are some reports indicating the role of rprA in antibiotic resistance. However, the role of rprA in ampicillin resistance is remained to be unknown. The aim of this study was to analyze the up regulation and down regulation of rprA and check their effects on ampicillin resistance in Escherichia coli. It was found that the up regulation and down regulation of rprA can lead into more antibiotics resistance and susceptibility, respectively. Our results showed the potential role of rprA expression in the response to ampicillin stress in E. coli.

Enterococcal bacteraemia: predictive and prognostic risk factors for ampicillin resistance.

To identify the predictive and prognostic factors associated with ampicillin-resistant enterococcal bacteraemia, we retrospectively reviewed demographic, microbiological and clinical data of patients attending the Kyoto University Hospital, Japan, between 2009 and 2015. Logistic regression and Cox regression analyses were performed to determine the predictive and prognostic factors, respectively. In total, 235 episodes of enterococcal bacteraemia were identified. As ampicillin susceptibility was uniform for Enterococcus faecalis isolates and almost all ampicillin-resistant isolates were E. faecium, bacteraemia due to these species was investigated separately. E. faecalis and E. faecium accounted for 41.7% (98/235) and 48.1% (113/235) of the isolates, respectively and 91.2% of all E. faecium were ampicillin resistant. Nosocomial E. faecium bacteraemia acquisition (odds ratio (OR), 13.6; 95% confidence intervals, 3.16-58.3) was associated with ampicillin-resistant isolates. Bacteraemia from an unknown source (hazard ratio (HR), 2.91; 95% CI 1.36-6.21) and an increased Pitt bacteraemia score (PBS) (HR, 1.36; 95% CI 1.21-1.52) were associated with 30-day mortality in E. faecium infections. Likewise, bacteraemia from an unknown source (HR, 4.17; 95% CI 1.25-13.9) and increased PBS (HR, 1.27; 95% CI 1.09-1.48) were associated with 30-day mortality in patients with E. faecalis bacteraemia. The empirical therapeutic administration of glycopeptides is recommended for patients with bacteraemia from an unknown source in whom severe E. faecium bacteraemia is suspected.

Imidazole decreases the ampicillin resistance of an Escherichia coli strain isolated from a cow with mastitis by inhibiting the function of autoinducer 2.

Extended-spectrum ß-lactamase-positive Escherichia coli is an important causative agent of mastitis in dairy cows that results in reduced milk production and quality, and is responsible for severe economic losses in the dairy industry worldwide. The quorum sensing signaling molecule autoinducer 2 (AI-2) is produced by many species of gram-negative and gram-positive bacteria, and might be a universal language for intraspecies and interspecies communication. Our previous work confirmed that exogenous AI-2 increases the antibiotic resistance of extended-spectrum ß-lactamase-positive E. coli to the ß-lactam group of antibiotics by upregulating the expression of the TEM-type ß-lactamase. In addition, this regulation relies on the function of the intracellular AI-2 receptor LsrR. In the present work, we reported that exogenous imidazole, a furan carbocyclic analog of AI-2, decreases the antibiotic resistance of a clinical E. coli strain to ß-lactam antibiotics by inhibiting the function of AI-2.

A single nucleotide in the promoter region modulates the expression of the ß-lactamase OXA-61 in Campylobacter jejuni.

OBJECTIVES: Despite prevalent ß-lactam resistance in Campylobacter jejuni, an important zoonotic enteric pathogen, the molecular basis of ß-lactamase-mediated ß-lactam resistance is still largely unknown. In particular, some C. jejuni strains that carry ß-lactamase gene blaOXA-61 (Cj0299) are still susceptible to ß-lactams with undetected ß-lactamase activity, suggesting blaOXA-61 is subjected to regulation. The objective of this study was to determine the regulatory mechanism of ß-lactamase in C. jejuni. METHODS: An ampicillin-resistant derivative of C. jejuni NCTC 11168 was subjected to whole genome sequencing and comparative genomics analysis. Complementary molecular experiments were further performed to examine the identified regulatory mechanism of blaOXA-61, which included complementation, promoter fusion assay, real-time RT-PCR, natural transformation using a defined PCR fragment, survey of clinical isolates and transcription start site mapping. RESULTS: A single nucleotide mutation (G → T transversion) upstream of blaOXA-61 was identified in the ampicillin-resistant derivative of NCTC 11168. The role of the G → T point mutation in acquired ß-lactam resistance through up-regulating the expression of blaOXA-61 was confirmed by multiple molecular approaches. The G → T transversion in the blaOXA-61 promoter was linked to high-level ß-lactam resistance in C. jejuni isolates. Transcription start site mapping indicated that the G → T transversion restored the TATA box in the -10 region of blaOXA-61. CONCLUSIONS: We demonstrated a novel genetic mechanism of ß-lactamase regulation in C. jejuni in this study, which will provide insights into the regulation and evolution of ß-lactam resistance in Campylobacter.

Human Ind1 expression causes over-expression of E. coli beta-lactamase ampicillin resistance protein.

Ind1, a mitochondrial P-loop NTPase is essential for assembly of respiratory complex-I. Respiratory complex-I (NADH: ubiquinone oxidoreductase), a large (mitochondrial inner membrane) enzyme, is made of 45 subunits and 8 iron-sulfur clusters. Ind1, an iron-sulfur cluster protein involved in the maturation of respiratory complex and binds an Fe/S cluster via a conserved CXXC motif in a labile way. Ind1 has been proposed as a specialized biogenesis factor involved in delivering the Fe/S clusters to the apo complex-I subunits. The IND1 gene is conserved in eukaryotes and is present in genomes of the species that retain functional respiratory complex-I. Depletion of human Ind1 causes ultra-structural changes in depleted mitochondria, including the loss of cristae membranes, massive remodeling of respiratory super complexes, and increased lactate production. Ind1 sequence bears known nucleotide binding domain motifs and was first classified as Nucleotide Binding Protein-Like (NUBPL). Despite the obvious importance of Ind1, very little is known about this protein; in particular its structure as well as its Fe/S cluster binding properties. In the present work we show that the expression of native huInd1 in Escherichia coli stimulates over-expression of the beta-lactamase TEM-1 from E. coli. The homology modeling of huInd1 shows hallmark of Rossmann fold, where a central beta sheet is covered by helices on either side. In the light of the modeled structure of huInd1, we hypothesize that huInd1 binds to the untranslated region (UTR) of the TEM-1 mRNA at 3' site and thereby reducing the possibility of its endonucleolytic cleavage, resulting in over-expression of TEM-1.

[Antibiotic resistance and virulence factors in clinical Salmonella enterica isolates]. / Resistencia a antibióticos y factores de virulencia en aislados clínicos de Salmonella enterica.

INTRODUCTION: The increase of Salmonella enterica isolates multi-resistant to different antibiotics, including ß-lactams and fluoroquinolones, is a problem of clinical importance. The dissemination of Salmonella Typhimurium resistant to ampicillin (AMP)-chloramphenicol (CHL)-streptomycin (STR)-sulphonamides and (SUL)-tetracycline (TET), that harbour the Salmonella Genomic Island type 1 (SGI1), and the acquisition of transferable genetic material have favoured the multi-resistance in this genus. METHODS: A total of 114 clinical S.enterica isolates were studied (period 2009-2010). The susceptibility to 20 antibiotics was determined by disc diffusion and microdilution. The antimicrobial resistance mechanisms and the integrons were analysed by PCR, and sequencing in the AMP(R) isolates. In all the blaPSE-1-positive isolates, the clonal relationship was determined by PFGE, as well as the presence of SGI1 and 29 virulence genes by PCR. RESULTS: Eighteen different serotypes were found among the 114 isolates studied, Typhimurium (61%) and Enteritidis (16%) being the most prevalent. High percentages of resistance to SUL (68%), TET (58%), AMP (55%) and STR (46%) were observed. The great majority (92%) of 63 AMP(R) isolates were multi-resistant, with the AMP-STR-TET-SUL phenotype (19 isolates) being the most frequent one and associated with the blaTEM-1b+strA-strB+tet(B)+sul2 genotype. Class 1 integrons (7 different structures) were observed in 48% AMP(R) isolates, highlighting the blaOXA-1+aadA1 structure (8 isolates), one empty integron and non-classical integrons (5 isolates). The blaPSE-1 gene was detected inside the classical SGI1 structure in 13 clonally-related isolates that showed the same virulence profile. CONCLUSIONS: The high percentage of multi-resistant S.enterica isolates, especially associated to S.Typhimurium, to the AMP, STR, TET and SUL phenotype, and to the blaTEM-1b+strA-strB+tet(B)+sul2 genotype, shows an important risk of possible failures in the treatment of serious infections caused by this serotype.

Genomic context as well as sequence of both psr and penicillin-binding protein 5 contributes to ß-lactam resistance in Enterococcus faecium.

Penicillin-binding protein 5 (PBP5) of Enterococcus faecium (Efm) is vital for ampicillin resistance (AMP-R). We previously designated three forms of PBP5, namely, PBP5-S in Efm clade B strains [ampicillin susceptible (AMP-S)], PBP5-S/R (AMP-S or R), and PBP5-R (AMP-R) in clade A strains. Here, pbp5 deletion resulted in a marked reduction in AMP minimum inhibitory concentrations (MICs) to 0.01-0.09 µg/mL for clade B and 0.12-0.19 µg/mL for clade A strains; in situ complementation restored parental AMP MICs. Using D344SRF (lacking ftsW/psr/pbp5), constructs with ftsWA/psrA (from a clade A1 strain) cloned upstream of pbp5-S and pbp5-S/R alleles resulted in modest increases in MICs to 3-8 µg/mL, while high MICs (>64 µg/mL) were seen using pbp5 from A1 strains. Next, using ftsW ± psr from clade B and clade A/B and B/A hybrid constructs, the presence of psrB, even alone or in trans, resulted in much lower AMP MICs (3-8 µg/mL) than when psrA was present (MICs >64 µg/mL). qRT PCR showed relatively greater pbp5 expression (P = 0.007) with pbp5 cloned downstream of clade A1 ftsW/psr (MIC >128 µg/mL) vs when cloned downstream of clade B ftsW/psr (MIC 4-16 µg/mL), consistent with results in western blots. In conclusion, we report the effect of clade A vs B psr on AMP MICs as well as the impact of pbp5 alleles from different clades. While previously, Psr was not thought to contribute to AMP MICs in Efm, our results showed that the presence of psrB resulted in a major decrease in Efm AMP MICs. IMPORTANCE: The findings of this study shed light on ampicillin resistance in Enterococcus faecium clade A strains. They underscore the significance of alterations in the amino acid sequence of penicillin-binding protein 5 (PBP5) and the pivotal role of the psr region in PBP5 expression and ampicillin resistance. Notably, the presence of a full-length psrB leads to reduced PBP5 expression and lower minimum inhibitory concentrations (MICs) of ampicillin compared to the presence of a shorter psrA, regardless of the pbp5 allele involved. Additionally, clade B E. faecium strains exhibit lower AMP MICs when both psr alleles from clades A and B are present, although it is important to consider other distinctions between clade A and B strains that may contribute to this effect. It is intriguing to note that the divergence between clade A and clade B E. faecium and the subsequent evolution of heightened AMP MICs in hospital-associated strains appear to coincide with changes in Pbp5 and psr. These changes in psr may have resulted in an inactive Psr, facilitating increased PBP5 expression and greater ampicillin resistance. These results raise the possibility that a mimicker of PsrB, if one could be designed, might be able to lower MICs of ampicillin-resistant E. faecium, thus potentially resorting ampicillin to our therapeutic armamentarium for this species.

PCR screening for the N526K substitution in isolates of Haemophilus influenzae and Haemophilus haemolyticus.

OBJECTIVES: Firstly, to evaluate the current PBP3-S primers of Hasegawa et al. (Microb Drug Resist 2003; 9: 39-46) and develop new primers for the amplification of N526 in isolates of Haemophilus haemolyticus. Secondly, to develop a new PCR assay for the detection (by amplification) of the N526K substitution, encoded by either the AAA or AAG single nucleotide polymorphism (SNP) at position 1576-1578 of the ftsI gene, in isolates of both Haemophilus influenzae and H. haemolyticus. METHODS: A total of 50 H. influenzae and 50 H. haemolyticus isolates, comprising N526 and N526K genotypes, were used to evaluate the performance of SNP-based PCR primers for the detection of the ß-lactamase-negative ampicillin resistance (BLNAR)-defining N526K substitution in H. influenzae and H. haemolyticus, using a real-time PCR platform. RESULTS: The PBP3-S primers of Hasegawa et al. failed to amplify H. haemolyticus isolates, irrespective of their N526/N526K status, owing to an inability of the forward primer to bind the H. haemolyticus ftsI sequence, giving an overall sensitivity of 100% and a specificity of 40% when using all of the isolates. However, the PBP3-N526 and PBP3-N526K PCR primers designed in this study were 100% sensitive and specific, and 84% sensitive and 100% specific, respectively, for the detection of N526K-positive isolates. CONCLUSIONS: Although antibiotic resistance surveillance studies on H. influenzae should include a definitive test for H. influenzae/H. haemolyticus identification, the new primers from this study will not only allow for PCR characterization of both H. influenzae and H. haemolyticus with respect to the N526K BLNAR substitutions, they will also stop incorrect characterization of susceptible H. haemolyticus isolates as low-BLNAR H. influenzae.

Escherichia coli from Human Wounds: Analysis of Resistance to ß-Lactams and Expression of RND Efflux Pumps.

Purpose: Resistance of pathogenic strains of Escherichia coli to ß-lactams, particularly to ampicillin, is on the rise and it is attributed to intrinsic and acquired mechanisms. One important factor contributing to resistance, together with primarily resistance mechanisms, is a mutation and/or an over-expression of the intrinsic efflux pumps in the resistance-nodulation-division (RND) superfamily. Among these efflux pumps, AcrA, AcrB, TolC, and AcrD play an important role in antimicrobial co-resistance, including resistance to ß-lactams. Materials and Methods: Twelve E. coli isolates obtained from patients' wounds and the control strain of E. coli ATCC 25922 were analyzed. The phenotypic resistance of these isolates to selected ß-lactams was assessed by determination of the minimal inhibitory concentration. Additionally, the prevalence of ß-lactamase genes (blaTEM, blaCTX-M, blaSHV, and blaAmpC) was screened by PCR. Real-time qPCR was used to determine the expression of the selected efflux pumps acrA, acrB, tolC, and acrD and the repressor acrR after the exposure of E. coli to ampicillin. Results: Phenotypic resistance to ß-lactams was detected in seven isolates, mainly to ampicillin and piperacillin. This was corroborated by the presence of at least one acquired bla gene in each of these isolates. Although E. coli strains varied in the expression of RND-family efflux pumps after the ampicillin exposure, their gene expression indicated that these pumps did not play a major role in the phenotypic resistance to ampicillin. Conclusion: Each E. coli isolate displayed unique characteristics, differing in minimum inhibitory concentration (MIC) values, prevalence of acquired blaTEM and blaCTX-M genes, and expression of the RND-family pumps. This together demonstrates that these clinical isolates employed distinct intrinsic or acquired resistance pathways for their defense against ampicillin. The prevalence and spread of ampicillin resistant E. coli has to be monitored and the search for ampicillin alternatives is needed.

Ampicillin and Gentamicin Treatment for Early Onset Neonatal Sepsis: When One Size Does Not Fit All.

Based on in vitro susceptibilities and the concern for emergence of resistance and long-term safety, ampicillin plus gentamicin remains the recommended antibiotic regimen for early onset neonatal sepsis. Our objective was to identify potential limitations of this regimen based on clinical and pathogen characteristics while minimizing risks associated with prolonged antibiotic exposure. We identified 43 gram-negative pathogens in 42 patients. Escherichia coli (E coli) occurred in 50% and Streptococcus agalactiae in 23.8% of patient. Ampicillin resistance was common, particularly in E coli (85.7%). Mortality was 23.8%, all due to E coli. We found that E coli is the most frequent pathogen and has a high mortality particularly in neonates < 1500 g; mortality is high with the current dosing strategy when E coli is resistant to ampicillin even when sensitive to gentamicin; resistance to gentamicin remains low but seems to be increasing while resistance to third-generation cephalosporins remains very low.

Efficient traceless modification of the P1 bacteriophage genome through homologous recombination with enrichment in double recombinants: A new perspective on the functional annotation of uncharacterized phage genes.

The advent of high-throughput omic technologies has caused unprecedented progress in research on bacteriophages, the most abundant and still the least explored entities on earth. Despite the growing number of phage genomes sequenced and the rejuvenation of interest in phage therapy, the progress in the functional analysis of phage genes is slow. Simple and efficient techniques of phage genome targeted mutagenesis that would allow one to knock out particular genes precisely without polar effects in order to study the effect of these knock-outs on phage functions are lacking. Even in the case of model phages, the functions of approximately half of their genes are unknown. P1 is an enterobacterial temperate myophage of clinical significance, which lysogenizes cells as a plasmid. It has a long history of studies, serves as a model in basic research, is a gene transfer vector, and is a source of genetic tools. Its gene products have structural homologs in several other phages. In this perspective article, we describe a simple and efficient procedure of traceless P1 genome modification that could also serve to acquire targeted mutations in the genomes of certain other temperate phages and speed up functional annotations of phage genes.

Catharanthus roseus (L.) G. Don counteracts the ampicillin resistance in multiple antibiotic-resistant Staphylococcus aureus by downregulation of PBP2a synthesis.

It is essential to revisit the global biodiversity, search for ethnopharmacologically relevant plants, and unveil their untapped potential to overcome the complications associated while treating infections triggered by multiple antibiotic-resistant Staphylococcus aureus. Catharanthus roseus (L.) G. Don of the Apocynaceae family is a medicinal plant used for remedial purposes against infectious diseases from ancient times. In this study, we intended to evaluate the mechanism by which the ethanolic extract of C. roseus root (EECRR) causes the reversal of ampicillin resistance in S. aureus. To achieve this goal, we have stained EECRR-treated S. aureus with acridine orange, analysed DNA damage by comet assay, and studied the alteration of plasmid band pattern and expression of penicillin-binding protein 2a (PBP2a) protein. Experiments revealed better S. aureus killing efficiency of EECRR at its minimum inhibitory concentration (MIC) doses due to DNA damage and reducing plasmid band intensities along with a decline in the expression of PBP2a in EECRR-treated cells at half-MIC dose. EECRR proved to be an efficient growth inhibitor of S. aureus that reduces the expression of PBP2a. Therefore, EECRR can also render ampicillin-resistant S. aureus susceptible to the antibiotic.

High-Resolution Genotyping Unveils Identical Ampicillin-Resistant Enterococcus faecium Strains in Different Sources and Countries: A One Health Approach.

Multidrug-resistant (MDR) Enterococcus faecium (Efm) infections continue to increase worldwide, although epidemiological studies remain scarce in lower middle-income countries. We aimed to explore which strains circulate in E. faecium causing human infections in Tunisian healthcare institutions in order to compare them with strains from non-human sources of the same country and finally to position them within the global E. faecium epidemiology by genomic analysis. Antibiotic susceptibility testing was performed and transfer of vancomycin-vanA and ampicillin-pbp5 resistance was performed by conjugation. WGS-Illumina was performed on Tunisian strains, and these genomes were compared with Efm genomes from other regions present in the GenBank/NCBI database (n = 10,701 Efm genomes available May 2021). A comparison of phenotypes with those predicted by the recent ResFinder 4.1-CGE webtool unveiled a concordance of 88%, with discordant cases being discussed. cgMLST revealed three clusters [ST18/CT222 (n = 13), ST17/CT948 strains (n = 6), and ST203/CT184 (n = 3)], including isolates from clinical, healthy-human, retail meat, and/or environmental sources in different countries over large time spans (10-12 years). Isolates within each cluster showed similar antibiotic resistance, bacteriocin, and virulence genetic patterns. pbp5-AmpR was transferred by VanA-AmpR-ST80 (clinical) and AmpR-ST17-Efm (bovine meat). Identical chromosomal pbp5-platforms carrying metabolic/virulence genes were identified between ST17/ST18 strains of clinical, farm animal, and retail meat sources. The overall results emphasize the role of high-resolution genotyping as provided by WGS in depicting the dispersal of MDR-Efm strains carrying relevant adaptive traits across different hosts/regions and the need of a One Health task force to curtail their spread.

Haemophilus influenzae type f in the post-Haemophilus influenzae type b vaccination era: a systematic review.

Since the introduction of Haemophilus influenzae (Hi) serotype b (Hib) vaccination, reports of increasing incidence rates of non-Hib serotypes have emerged. A systematic review was performed to investigate whether the Hi serotype f (Hif) incidence rate has increased globally and to describe its associated disease burden. In the post-Hib vaccine era, evidence shows that the incidence rate of Hif infection is increasing worldwide. In total 94 studies including 2 701 patients reported Hif infections. The estimated pooled incidence rate of Hif infection was 0.15/100 000 population per year (range: 0.05-0.40/100 000), with a median case fatality ratio of 14.3 %. Invasive infections most frequently presented as pneumonia (45 %), septicaemia (34 %) and meningitis (20 %). Of 191 Hif isolates, 87 % were ampicillin-susceptible. Multi-locus sequence typing revealed that Hif were relatively clonal, with the majority belonging to clonal complex 124. Hif causes invasive infections of significant variance in both severity and presentation. Globally, the Hif population shows little genetic variability and currently appears to possess low resistance to antimicrobials.

Antimicrobial Resistance of Acetobacter and Komagataeibacter Species Originating from Vinegars.

Consumers' preference towards healthy and novel foods dictates the production of organic unfiltered bottled vinegar that still contains acetic acid bacteria. After ingesting vinegar, the bacteria come into close contact with the human microbiota, creating the possibility of horizontal gene transfer, including genetic determinants for antibiotic resistance. Due to the global spread of antimicrobial resistance (AMR), we analyzed the AMR of Acetobacter and Komagataeibacter species originating mainly from vinegars. Six antibiotics from different structural groups and mechanisms of action were selected for testing. The AMR was assessed with the disk diffusion method using various growth media. Although the number of resistant strains differed among the growth media, 97.4%, 74.4%, 56.4%, and 33.3% of strains were resistant to trimethoprim, erythromycin, ciprofloxacin, and chloramphenicol, respectively, on all three media. Moreover, 17.9% and 53.8% of all strains were resistant to four and three antibiotics of different antimicrobial classes, respectively. We then looked for antimicrobial resistance genes in the genome sequences of the reference strains. The most common genetic determinant potentially involved in AMR encodes an efflux pump. Since these genes pass through the gastrointestinal tract and may be transferred to human microbiota, further experiments are needed to analyze the probability of this scenario in more detail.