Β-lactam Resistance Research Articles

A strategy design based on antibiotic‑resistance and plasmid replicons genes of clinical Escherichia coli strains

ABSTRACT Since antimicrobial resistance, especially β-lactam resistance genes were common in clinical Escherichia coli strains, this study had designed and developed multiplex amplification platform for rapid and accurate detection of such resistance genes in 542 clinical E. coli isolates. The obtained specimens were subjected to bacteriological examination, antimicrobial susceptibility testing, and detection of β-lactamase genes and plasmid replicons. The major virulence genes were detected by 7 groups of multiplex PCR and eight groups of multiplex PCR were designed to detect 8 different plasmid replicons including parA-parB, iteron, repA, and RNAI. It was found that most MDR isolates were co-resistant to penicillins (AMP) and fluoroquindones (LVX, CIP) and distribution of LVX and CIP resistance was significantly higher among female than male gender. RNAI (AY234375) showed the highest detection rate, followed by the iteron (J01724) and repA (M26308), indicating the relatively higher carriage rate of corresponding plasmids. Bla OXA acquired the highest carriage rate, followed by group 2 bla CTX-M and bla SHV-1, indicating their prevalence among clinical E. coli. Among the β-lactamase genes, bla OXA acquired the highest carriage rate, followed by group 2 bla CTX-M and bla SHV-1, indicating their prevalence among clinical E. coli. The RNAI (AY234375) showed the highest detection rate, followed by the iteron (J01724) and repA (M26308), indicating the relatively higher carriage rate of the corresponding plasmids by clinical E. coli isolates. It is shown that the developed multiplex amplification methodology is applicable to AMR detection, and such identification of plasmid replicons and β-lactamase genes may aid in the understanding of clinical E. coli isolate epidemiology.

Gut Commensal Escherichia coli, a High-Risk Reservoir of Transferable Plasmid-Mediated Antimicrobial Resistance Traits.

BackgroundEscherichia coli (E. coli), the main human gut microorganism, is one of the evolved superbugs because of acquiring antimicrobial resistance (AMR) determinants via horizontal gene transfer (HGT).PurposeThis study aimed to screen isolates of gut commensal E. coli from healthy adult individuals for antimicrobial susceptibility and plasmid-mediated AMR encoding genes.MethodsGut commensal E. coli bacteria were isolated from fecal samples that were taken from healthy adult individuals and investigated phenotypically for their antimicrobial susceptibility against diverse classes of antimicrobials using the Kirby Bauer disc method. PCR-based molecular assays were carried out to detect diverse plasmid-carried AMR encoding genes and virulence genes of different E. coli pathotypes (eaeA, stx, ipaH, est, elt, aggR and pCVD432). The examined AMR genes were β-lactam resistance encoding genes (blaCTX-M1, blaTEM, blaCMY-2), tetracycline resistance encoding genes (tetA, tetB), sulfonamides resistance encoding genes (sul1, sulII), aminoglycoside resistance encoding genes (aac(3)-II, aac(6′)-Ib-cr) and quinolones resistance encoding genes (qnrA, qnrB, qnrS).ResultsPCR results revealed the absence of pathotypes genes in 56 isolates that were considered gut commensal isolates. E. coli isolates showed high resistance rates against tested antimicrobial agents belonging to both β-lactams and sulfonamides (42/56, 75%) followed by quinolones (35/56, 62.5%), tetracyclines (31/56, 55.4%), while the lowest resistance rate was to aminoglycosides (24/56, 42.9%). Antimicrobial susceptibility profiles revealed that 64.3% of isolates were multidrug-resistant (MDR). High prevalence frequencies of plasmid-carried AMR genes were detected including blaTEM (64%) sulI (60.7%), qnrA (51.8%), aac(3)-II (37.5%), and tetA (46.4%). All isolates harbored more than one gene with the most frequent genetic profile among isolates was blaTEM-blaCTX-M1-like-qnrA-qnrB-tetA-sulI.ConclusionResults are significant in the evaluation of plasmid-carried AMR genes in the human gut commensal E. coli, suggesting a potential human health risk and the necessity of strict regulation of the use of antibiotics in Egypt. Commensal E. coli bacteria may constitute a potential reservoir of AMR genes that can be transferred to other bacterial species.

Impact of polymorphisms in blaZ, blaR1 and blaI genes and their relationship with β-lactam resistance in S. aureus strains isolated from bovine mastitis

There is not a consensus between the presence of the genotypic resistance marker gene and the phenotypic resistance to β-lactams in Staphylococcus aureus, which means, positive S. aureus blaZ isolates demonstrating sensitivity to β-lactams. The present study aimed to characterize the blaZ, blaR1 and blaI genes, identify and evaluate single nucleotide polymorphisms (SNPs) and their relationship with β-lactam resistance in samples of Staphylococcus aureus obtained from cases of bovine mastitis. Five isolates (two resistant and three sensitive to oxacillin) of Staphylococcus aureus with detected production of beta-lactamase, previously evaluated as containing the blaZ gene and negative for the mecA and mecC genes, had the bla operon completely sequenced. Impacts on the protein sequence due to the detected polymorphisms were evaluated by modeling the proteins encoded by the blaZ, blaR1 and blaI genes using a three-dimensional model structure obtained from the Protein Data Bank (PDB) database. Fifteen SNPs were detected in the blaZ gene, 30 in the blaR1 gene and three in the blaI gene. These SNPs caused alterations in amino acid sites. Deleterious mutations were detected in the blaZ gene (E146G, P218S, Y221C) and the blaR1 gene (K481E). Molecular docking analysis revealed that polymorphisms in the blaZ gene may explain the phenotypic sensitivity in isolates that contain the resistance marker gene. Although sensitive and resistant isolates encode beta-lactamase, these proteins are functionally altered due to a change in the binding site with the antibiotic.

Signal Peptidase-Mediated Cleavage of the Anti-σ Factor RsiP at Site 1 Controls σP Activation and β-Lactam Resistance in Bacillus thuringiensis.

ABSTRACTIn Bacillus thuringiensis, β-lactam antibiotic resistance is controlled by the extracytoplasmic function (ECF) σ factor σP. σP activity is inhibited by the anti-σ factor RsiP. In the presence of β-lactam antibiotics, RsiP is degraded and σP is activated. Previous work found that RsiP degradation requires cleavage of RsiP at site 1 by an unknown protease, followed by cleavage at site 2 by the site 2 protease RasP. The penicillin-binding protein PbpP acts as a sensor for β-lactams. PbpP initiates σP activation and is required for site 1 cleavage of RsiP but is not the site 1 protease. Here, we describe the identification of a signal peptidase, SipP, which cleaves RsiP at a site 1 signal peptidase cleavage site and is required for σP activation. Finally, many B. anthracis strains are sensitive to β-lactams yet encode the σP-RsiP signal transduction system. We identified a naturally occurring mutation in the signal peptidase cleavage site of B. anthracis RsiP that renders it resistant to SipP cleavage. We find that B. anthracis RsiP is not degraded in the presence of β-lactams. Altering the B. anthracis RsiP site 1 cleavage site by a single residue to resemble B. thuringiensis RsiP results in β-lactam-dependent degradation of RsiP. We show that mutation of the B. thuringiensis RsiP cleavage site to resemble the sequence of B. anthracis RsiP blocks degradation by SipP. The change in the cleavage site likely explains many reasons why B. anthracis strains are sensitive to β-lactams.

P26 Phenotypic and genotypic characterization of antimicrobial resistance in Clostridioides difficile in North Wales

Background Clostridioides difficile poses a major infection control challenge and remains a huge burden to our healthcare system. The emergence of hypervirulent strains of C. difficile has highlighted the need for monitoring antimicrobial resistance (AMR) and continual epidemiological vigilance. Agar dilution is the current gold standard method for antimicrobial susceptibility testing of C. difficile and the method routinely utilized in the Public Health Wales (PHW) UK Anaerobe Reference Unit (UKARU) at the University Hospital of Wales, Cardiff. However, this method is laborious and time consuming. Meanwhile, the emergence of WGS introduces the possibility of acquiring rapid genotypic information in terms of AMR.MethodsIn this research project, isolates obtained from faecal samples submitted across the Betsi Cadwalader health board (BCUHB) that were either GDH or PCR positive were sequenced as part of the C. difficile Genomic Sequencing and Typing (DIGEST) pilot project performed at the PHW Pathogen Genomics Unit (PenGU) in Cardiff.ResultsResults highlights include the finding that 93% of the isolates analysed for the presence of AMR genes were found to have the cdeA gene associated with conferring resistance to fluoroquinolones such as ciprofloxacin. Other common genes observed included blaCDD-1 and blaCDD-2, associated with β-lactam resistance.ConclusionsWhile further work must be done to link genotypic characterization with phenotype, this study provides a valuable insight into AMR mechanisms in C. difficile and may in future become an important tool providing early indication of shift in C. difficile epidemiology or the emergence of a new hypervirulent and MDR strain.

Impact of Peptidoglycan Recycling Blockade and Expression of Horizontally Acquired β-Lactamases on Pseudomonas aeruginosa Virulence.

ABSTRACTIn the current scenario of antibiotic resistance magnification, new weapons against top nosocomial pathogens like Pseudomonas aeruginosa are urgently needed. The interplay between β-lactam resistance and virulence is considered a promising source of targets to be attacked by antivirulence therapies, and in this regard, we previously showed that a peptidoglycan recycling blockade dramatically attenuated the pathogenic power of P. aeruginosa strains hyperproducing the chromosomal β-lactamase AmpC. Here, we sought to ascertain whether this observation could be applicable to other β-lactamases. To do so, P. aeruginosa wild-type or peptidoglycan recycling-defective strains (ΔampG and ΔnagZ) harboring different cloned β-lactamases (transferable GES, VIM, and OXA types) were used to assess their virulence in Galleria mellonella larvae by determining 50% lethal doses (LD50s). A mild yet significant LD50 increase was observed after peptidoglycan recycling disruption per se, whereas the expression of class A and B enzymes did not impact virulence. While the production of the narrow-spectrum class D OXA-2 entailed a slight attenuation, its extended-spectrum derivatives OXA-226 (W159R [bearing a change of W to R at position 159]), OXA-161 (N148D), and principally, OXA-539 (D149 duplication) were associated with outstanding virulence impairments, especially in recycling-defective backgrounds (with some LD50s being >1,000-fold that of the wild type). Although their exact molecular bases remain to be deciphered, these results suggest that mutations affecting the catalytic center and, therefore, the hydrolytic spectrum of OXA-2-derived enzymes also drastically impact the pathogenic power of P. aeruginosa. This work provides new and relevant knowledge to the complex topic of the interplay between the production of β-lactamases and virulence that could be useful to build future therapeutic strategies against P. aeruginosa.IMPORTANCE Pseudomonas aeruginosa is one of the leading nosocomial pathogens whose growing resistance makes the development of therapeutic options extremely urgent. The resistance-virulence interplay has classically aroused researchers’ interest as a source of therapeutic targets. In this regard, we describe a wide array of virulence attenuations associated with different transferable β-lactamases, among which the production of OXA-2-derived extended-spectrum β-lactamases stood out as a dramatic handicap for pathogenesis, likely as a side effect of mutations causing the expansion of their hydrolytic spectrums. Moreover, our results confirm the validity of disturbing peptidoglycan recycling as a weapon to attenuate P. aeruginosa virulence in class C and D β-lactamase production backgrounds. In the current scenario of dissemination of horizontally acquired β-lactamases, this work brings out new data on the complex interplay between the production of specific enzymes and virulence attenuation that, if complemented with the characterization of the underlying mechanisms, will likely be exploitable to develop future virulence-targeting antipseudomonal strategies.

Rapid Inhibitor Discovery by Exploiting Synthetic Lethality.

Synthetic lethality occurs when inactivation of two genes is lethal but inactivation of either single gene is not. This phenomenon provides an opportunity for efficient compound discovery. Using differential growth screens, one can identify biologically active compounds that selectively inhibit proteins within the synthetic lethal network of any inactivated gene. Here, based purely on synthetic lethalities, we identified two compounds as the only possible inhibitors of Staphylococcus aureus lipoteichoic acid (LTA) biosynthesis from a screen of ∼230,000 compounds. Both compounds proved to inhibit the glycosyltransferase UgtP, which assembles the LTA glycolipid anchor. UgtP is required for β-lactam resistance in methicillin-resistant S. aureus (MRSA), and the inhibitors restored sensitivity to oxacillin in a highly resistant S. aureus strain. As no other compounds were pursued as possible LTA glycolipid assembly inhibitors, this work demonstrates the extraordinary efficiency of screens that exploit synthetic lethality to discover compounds that target specified pathways. The general approach should be applicable not only to other bacteria but also to eukaryotic cells.

Whole Genome Analysis of 335 New Bacterial Species from Human Microbiota Reveals a Huge Reservoir of Transferable Antibiotic Resistance Determinants.

Background: The emergence and diffusion of strains of pathogenic bacteria resistant to antibiotics constitutes a real public health challenge. Antibiotic resistance genes (ARGs) can be carried by both pathogenic and non-pathogenic bacteria, including commensal bacteria from the human microbiota, which require special monitoring in the fight against antimicrobial resistance. Methods: We analyzed the proteomes of 335 new bacterial species from human microbiota to estimate its whole range of ARGs using the BLAST program against ARGs reference databases. Results: We found 278 bacteria that harbor a total of 883 potential ARGs with the following distribution: 264 macrolides-lincosamides-streptogramin, 195 aminoglycosides, 156 tetracyclines, 58 β-lactamases, 58 fosfomycin, 51 glycopeptides, 36 nitroimidazoles, 33 phenicols and 32 rifamycin. Furthermore, evolutionary analyses revealed the potential horizontal transfer with pathogenic bacteria involving mobile genetic elements such as transposase and plasmid. We identified many ARGs that may represent new variants in fosfomycin and β-lactams resistance. Conclusion: These findings show that new bacterial species from human microbiota should be considered as an important reservoir of ARGs that can be transferred to pathogenic bacteria. In vitro analyses of their phenotypic potential are required to improve our understanding of the functional role of this bacterial community in the development of antibiotic resistance.

Induction of Broad β-lactam Resistance in Achromobacter ruhlandii by Exposure to Ticarcillin Is Primarily Linked to Substitutions in Murein Peptide Ligase Mpl.

Achromobacter species are emerging pathogens in cystic fibrosis with inherent resistance to several classes of antimicrobial agents. We exposed strains with wild-type antimicrobial susceptibility to ticarcillin and generated mutants with broad β-lactam resistance. Within the detection limit of the assay, the capability to develop mutational resistance was strain-specific and reproducible. Mutational resistance was observed for all three tested strains of Achromobacter ruhlandii, for one of seven strains of Achromobacter xylosoxidans, and for none of five strains of Achromobacter insuavis. All mutants were resistant to piperacillin-tazobactam, while minimal inhibitory concentration of several other β-lactams increased 4–32-fold. Whole genome sequencing identified 1–4 non-synonymous mutations in known genes per mutant. All mutants encoded amino acid substitutions in cell wall recycling proteins, primarily Mpl, and the observed resistance is probably caused by hyperproduction of OXA-114-like β-lactamases. Related, but not identical substitutions were detected in clinical strains expressing acquired antimicrobial resistance.

The Prevalence and Molecular Biology of Staphylococcus aureus Isolated from Healthy and Diseased Equine Eyes in Egypt

This work aimed to characterize S. aureus isolates from the eyes of healthy and clinically affected equines in the Kafrelsheikh Governorate, Egypt. A total of 110 animals were examined for the presence of S. aureus, which was isolated from 33 animals with ophthalmic lesions and 77 healthy animals. We also investigated the antimicrobial resistance profile, oxacillin resistance mechanism, and the major virulence factors implicated in many studies of the ocular pathology of pathogenic S. aureus. The association between S. aureus eye infections and potential risk factors was also investigated. The frequency of S. aureus isolates from clinically affected equine eyes was significantly higher than in clinically healthy equids. A significant association was found between the frequency of S. aureus isolation from clinically affected equine eyes and risk factors including age and season but not with sex or breed factors. Antimicrobial resistance to common antibiotics used to treat equine eyes was also tested. Overall, the isolates showed the highest sensitivity to sulfamethoxazole (100%) and the highest resistance to cephalosporin (90.67%) and oxacillin (90.48%). PCR was used to demonstrate that mecA was present in 100% of oxacillin- and β-lactam-resistant S. aureus strains. The virulence factor genes Spa (x region), nuc, and hlg were identified in 62.5%, 100%, and 56%, of isolates, respectively, from clinically affected equines eyes. The severity of the eye lesions increased in the presence of γ-toxin-positive S. aureus. The phylogenetic tree of the Spa (x region) gene indicated a relationship with human reference strains isolated from Egypt as well as isolates from equines in Iran and Japan. This study provides insight into the prevalence, potential risk factors, clinical pictures, zoonotic potential, antimicrobial resistance, and β-lactam resistance mechanism of S. aureus strains that cause eye infection in equines from Egypt.

Genomic analysis reveals high intra-species diversity of Shewanella algae.

Shewanella algae is widely distributed in marine and freshwater habitats, and has been proved to be an emerging marine zoonotic and human pathogen. However, the genomic characteristics and pathogenicity of Shewanella algae are unclear. Here, the whole-genome features of 55 S . algae strains isolated from different sources were described. Pan-genome analysis yielded 2863 (19.4 %) genes shared among all strains. Functional annotation of the core genome showed that the main functions are focused on basic lifestyle such as metabolism and energy production. Meanwhile, the phylogenetic tree of the single nucleotide polymorphisms (SNPs) of core genome divided the 55 strains into three clades, with the majority of strains from China falling into the first two clades. As for the accessory genome, 167 genomic islands (GIs) and 65 phage-related elements were detected. The CRISPR-Cas system with a high degree of confidence was predicted in 23 strains. The GIs carried a suite of virulence genes and mobile genetic elements, while prophages contained several transposases and integrases. Horizontal genes transfer based on homology analysis indicated that these GIs and prophages were parts of major drivers for the evolution and the environmental adaptation of S. algae . In addition, a rich putative virulence-associated gene pool was found. Eight classes of antibiotic-associated resistance genes were detected, and the carriage rate of β-lactam resistance genes was 100 %. In conclusion, S. algae exhibits a high intra-species diversity in the aspects of population structure, virulence-associated genes and potential drug resistance, which is helpful for its evolution in pathogenesis and environmental adaptability.

Identification and Evaluation of Brominated Carbazoles as a Novel Antibiotic Adjuvant Scaffold in MRSA.

Antibiotic-resistant infections are a pressing global concern, causing millions of deaths each year. Methicillin-resistant Staphylococcus aureus (MRSA) is a leading cause of nosocomial infections in healthcare settings and is increasingly responsible for community-acquired infections that are often more difficult to treat. Antibiotic adjuvants are small molecules that potentiate antibiotics through nontoxic mechanisms and show excellent promise as novel therapeutics. Screening of low-molecular-weight compounds was employed to identify novel antibiotic adjuvant scaffolds for further elaboration. Brominated carbazoles emerged from this screening as lead compounds for further evaluation. Lead carbazoles were able to potentiate several β-lactam antibiotics in three medically relevant strains of MRSA. Gene expression studies determined that these carbazoles were dampening the transcription of key genes that modulate β-lactam resistance in MRSA. The lead brominated carbazoles represent novel scaffolds for elaboration as antibiotic adjuvants.

Activity of cefepime/taniborbactam and comparators against whole genome sequenced ertapenem-non-susceptible Enterobacterales clinical isolates: CANWARD 2007-19.

ObjectivesThis study assessed in vitro activities of cefepime/taniborbactam and comparator antimicrobial agents against ertapenem-non-susceptible Enterobacterales (ENSE) clinical isolates collected from the CANWARD study 2007–19, and associations between MIC and various mechanisms of β-lactam resistance identified using WGS.MethodsA total of 179 ENSE (MIC ≥ 1 mg/L) isolates underwent susceptibility testing using reference CLSI broth microdilution. WGS was performed using the Illumina NextSeq platform. Carbapenemases, ESBLs and other β-lactamases were identified using ResFinder 4.0. Alterations in ompC/F and ftsI (PBP3) were identified by comparing extracted sequences to the appropriate NCBI reference gene. Porin alterations were analysed with Provean v1.1.3. Specific alterations of interest in PBP3 included a YRIN or YRIK insertion after P333.ResultsCefepime/taniborbactam was highly active (MIC50/MIC90, 0.5/2 mg/L; 177/179 isolates inhibited at ≤ 8 mg/L) against ENSE with various antimicrobial resistance phenotypes. Thirteen (7.3%) of the 179 ENSE isolates demonstrated cefepime/taniborbactam MIC values ≥ 4 mg/L and possessed combinations of β-lactam resistance mechanisms, including a carbapenemase and/or ESBL and/or other β-lactamase genes, as well as alterations in OmpC and/or OmpF and/or PBP3. Of the two Escherichia coli isolates that demonstrated a cefepime/taniborbactam MIC of 32 mg/L, one possessed NDM-5, OXA-181 and TEM-1B, an OmpC alteration and P333_Y334insYRIN in PBP3, while the second contained CTX-M-71, a truncated OmpF and a large alteration in OmpC (F182_R195delinsMTTNGRDDVFE).ConclusionsCefepime/taniborbactam was highly active against ENSE with various antimicrobial resistance phenotypes/genotypes. ENSE isolates with cefepime/taniborbactam MIC values ≥ 4 mg/L possessed combinations of β-lactam resistance mechanisms, including β-lactamase genes, as well as alterations in OmpC and/or OmpF and/or PBP3.

Emergence of Meropenem Resistance Among Cefotaxime Non-susceptible Streptococcus pneumoniae: Evidence and Challenges

The principal causative agent of acute bacterial meningitis (ABM) in children and the elderly is Streptococcus pneumoniae, with a widespread increase in penicillin resistance. Resistance is due to non-synonymous single-nucleotide polymorphisms (nsSNPs) that alter the penicillin-binding proteins (PBPs), the targets for all β-lactam drugs. Hence, resistance against one β-lactam antibiotic may positively select another. Since meropenem is an alternative to cefotaxime in meningeal infections, we aim to identify whether nsSNPs in the PBPs causing penicillin and cefotaxime resistance can decrease the pneumococcal susceptibility to meropenem. Comparison of the nsSNPs in the PBPs between the cefotaxime-resistant Indian (n = 33) and global isolates (n = 28) revealed that nsSNPs in PBP1A alone elevated meropenem minimal inhibitory concentrations (MICs) to 0.12 μg/ml, and nsSNPs in both PBP2X and 2B combined with PBP1A increases MIC to ≥ 0.25 μg/ml. Molecular docking confirmed the decrease in the PBP drug binding affinity due to the nsSNPs, thereby increasing the inhibition potential and the MIC values, leading to resistance. Structural dynamics and thermodynamic stability pattern in PBPs as a result of mutations further depicted that the accumulation of certain nsSNPs in the functional domains reduced the drug affinity without majorly affecting the overall stability of the proteins. Restricting meropenem usage and promoting combination therapy with antibiotics having non-PBPs as targets to treat cefotaxime non-susceptible S. pneumoniae meningitis can prevent the selection of β-lactam resistance.

The role of Musca domestica and milk in transmitting pathogenic multidrug-resistant Escherichia coli and associated phylogroups to neonatal calves.

Escherichia coli, as a global source of antimicrobial resistance, is a serious veterinary and public health concern. The transmission of pathogenic multidrug-resistant (MDR) E. coli within diarrheic calves and its correlation with Musca domestica and milk strains have been investigated. In total, 110, 80, and 26 E. coli strains were obtained from 70 rectal swabs from diarrheic calves, 60 milk samples and 20M. domestica, respectively. Molecular pathotyping of E. coli revealed the presence of pathogenic E. coli with a higher percentage of shigatoxigenic strains within diarrheic calves and M. domestica at 46.4% and 34.6%, respectively. Phenotypic antimicrobial resistance revealed higher β-lactams resistance except for cefquinome that exhibited low resistance in M.domestica and milk strains at 30.8% and 30%, respectively. The extended-spectrum cephalosporin (ESC) resistant strains were detected within fecal, M. domestica, and milk strains at 69.1%, 73.1%, and 71.3%, respectively. All E. coli strains isolated from M. domestica exhibited MDR, while fecal and milk strains were harboring MDR at 99.1% and 85%, respectively. Molecular detection of resistant genes revealed the predominance of the blaTEM gene, while none of these strains harbored the blaOXA gene. The highest percentages for blaCTXM and blaCMYII genes were detected in M. domestica strains at 53.8% and 61.5%, respectively. Regarding colistin resistance, the mcr-1 gene was detected only in fecal and milk strains at 35.5% and 15%, respectively. A high frequency of phylogroup B2 was detected within fecal and M. domestica strains, while milk strains were mainly assigned to the B1 phylogroup. Pathogenic E. coli strains with the same phenotypic and genotypic antimicrobial resistance and phylogroups were identified for both diarrheic calves and M. domestica, suggesting that the possible role of M. domestica in disseminating pathogenic strains and antimicrobial resistance in dairy farms.

Occurrence, antibiotic susceptibility and resistance genes among Staphylococcus aureus isolated from keypads of automated teller machines (ATM) in a private university, Nigeria

Environmental surfaces and objects associated with dermal contact by multiple users are suspects of being reservoirs of microorganisms; the ATM is likely to be one of such surfaces. Staphylococcus aureus is an opportunistic organism and causes infection. S. aureus is a major public health interest because of its antibiotic resistance attributes. Considering this fact, a study on the occurrence, susceptibility pattern, and resistant genes among S. aureus isolates from ATM keypads of three different banks located in a private university in Nigeria, was carried out. A total of 56 swabbed samples were collected from seven ATM keypads belonging to the three different banks, and cultured on Mannitol Salt Agar for the isolation of Staphylococcus aureus. Using disk diffusion technique, 0.5 McFarland of each isolate was screened for antibiotic susceptibility, on Mueller-Hinton agar. Also, six of the isolates were screened for genotypic characterization and the detection of β-lactam (blaZ) and macrolide (ermB) resistance genes. Out of 56 isolates obtained, 30 (54%) were phenotypically identified as Staphylococcus aureus using the standard biochemical test. The isolates were 100% resistant to augmentin, ceftazidime, ceftriaxone, cloxacillin and cefuroxime but were susceptible to gentamycin and ofloxacin. On the molecular level, four out of the six randomly selected isolates showed amplified base pair product for S. aureus, blaZ gene as well as the absence of the gene encoding ermB. The present result has shown the potential contamination of ATM keyboards with Staphylococcus aureus and the ATM keypads can also serve as prospective platforms for cross-contamination with antibiotic-resistant strains especially when frequently used by clients of affiliated financial institutions. Therefore, this implies that the hygiene practice of handwashing with soap and water after ATM usage should be further reinforced to reduce the level of contamination and spread of this pathogen.

Docking and Molecular Dynamic of Microalgae Compounds as Potential Inhibitors of Beta-Lactamase.

Bacterial resistance is responsible for a wide variety of health problems, both in children and adults. The persistence of symptoms and infections are mainly treated with β-lactam antibiotics. The increasing resistance to those antibiotics by bacterial pathogens generated the emergence of extended-spectrum β-lactamases (ESBLs), an actual public health problem. This is due to rapid mutations of bacteria when exposed to antibiotics. In this case, β-lactamases are enzymes used by bacteria to hydrolyze the beta-lactam rings present in the antibiotics. Therefore, it was necessary to explore novel molecules as potential β-lactamases inhibitors to find antibacterial compounds against infection caused by ESBLs. A computational methodology based on molecular docking and molecular dynamic simulations was used to find new microalgae metabolites inhibitors of β-lactamase. Six 3D β-lactamase proteins were selected, and the molecular docking revealed that the metabolites belonging to the same structural families, such as phenylacridine (4-Ph), quercetin (Qn), and cryptophycin (Cryp), exhibit a better binding score and binding energy than commercial clinical medicine β-lactamase inhibitors, such as clavulanic acid, sulbactam, and tazobactam. These results indicate that 4-Ph, Qn, and Cryp molecules, homologous from microalgae metabolites, could be used, likely as novel β-lactamase inhibitors or as structural templates for new in-silico pharmaceutical designs, with the possibility of combatting β-lactam resistance

Identification of the Extracytoplasmic Function σ Factor σP Regulon in Bacillus thuringiensis.

ABSTRACTBacillus thuringiensis and other members of the Bacillus cereus family are resistant to many β-lactams. Resistance is dependent upon the extracytoplasmic function sigma factor σP. We used label-free quantitative proteomics to identify proteins whose expression was dependent upon σP. We compared the protein profiles of strains which either lacked σP or overexpressed σP. We identified 8 members of the σP regulon which included four β-lactamases as well as three penicillin-binding proteins (PBPs). Using transcriptional reporters, we confirmed that these genes are induced by β-lactams in a σP-dependent manner. These genes were deleted individually or in various combinations to determine their role in resistance to a subset of β-lactams, including ampicillin, methicillin, cephalexin, and cephalothin. We found that different combinations of β-lactamases and PBPs are involved in resistance to different β-lactams. Our data show that B. thuringiensis utilizes a suite of enzymes to protect itself from β-lactam antibiotics.IMPORTANCE Antimicrobial resistance is major concern for public health. β-Lactams remain an important treatment option for many diseases. However, the spread of β-lactam resistance continues to rise. Many pathogens acquire antibiotic resistance from environmental bacteria. Thus, understanding β-lactam resistance in environmental strains may provide insights into additional mechanisms of antibiotic resistance. Here, we describe how a single regulatory system, σP, in B. thuringiensis controls expression of multiple genes involved in resistance to β-lactams. Our findings indicate that some of these genes are partially redundant. Our data also suggest that the large number of genes controlled by σP results in increased resistance to a wider range of β-lactam classes than any single gene could provide.

Three-Dimensional Structure and Optimization of the Metallo-β-Lactamase Inhibitor Aspergillomarasmine A.

The aminopolycarboxylic acid aspergillomarasmine A (AMA) is a natural Zn2+ metallophore and inhibitor of metallo-β-lactamases (MBLs) which reverses β-lactam resistance. The first crystal structure of an AMA coordination complex is reported and reveals a pentadentate ligand with distorted octahedral geometry. We report the solid-phase synthesis of 23 novel analogs of AMA involving structural diversification of each subunit (l-Asp, l-APA1, and l-APA2). Inhibitory activity was evaluated in vitro using five strains of Escherichia coli producing globally prevalent MBLs. Further in vitro assessment was performed with purified recombinant enzymes and intracellular accumulation studies. Highly constrained structure–activity relationships were demonstrated, but three analogs revealed favorable characteristics where either Zn2+ affinity or the binding mode to MBLs were improved. This study identifies compounds that can further be developed to produce more potent and broader-spectrum MBL inhibitors with improved pharmacodynamic/pharmacokinetic properties.

Seven-Year Evolution of β-Lactam Resistance Phenotypes in Escherichia coli Isolated from Young Diarrheic and Septicaemic Calves in Belgium.

Antimicrobial resistance is a major worldwide hazard. Therefore, the World Health Organization has proposed a classification of antimicrobials with respect to their importance for human medicine and advised some restriction of their use in veterinary medicine. In Belgium, this regulation has been implemented by a Royal Decree (RD) in 2016, which prohibits carbapenem use and enforces strict restrictions on the use of third- and fourth-generation cephalosporins (3 GC and 4 GC) for food-producing animals. Acquired resistance to β-lactam antibiotics is most frequently mediated by the production of β-lactamases in Gram-negative bacteria. This study follows the resistance to β-lactam antibiotics in Escherichia coli isolated from young diarrheic or septicaemic calves in Belgium over seven calving seasons in order to measure the impact of the RD. Phenotypic resistance to eight β-lactams was assessed by disk diffusion assay and isolates were assigned to four resistance profiles: narrow-spectrum β-lactamases (NSBL); extended-spectrum β-lactamases (ESBL); cephalosporinases (AmpC); and cephalosporinase-like, NSBL with cefoxitin resistance (AmpC-like). No carbapenemase-mediated resistance was detected. Different resistance rates were observed for each profile over the calving seasons. Following the RD, the number of susceptibility tests has increased, the resistance rate to 3 GC/4 GC has markedly decreased, while the observed resistance profiles have changed, with an increase in NSBL profiles in particular.