Antibacterial and Antibiofilm Activity of Mercaptophenol Functionalized-Gold Nanorods Against a Clinical Isolate of Methicillin-Resistant Staphylococcus Aureus

Gold nanorods (AuNRs) were synthesized and surface functionalized with 4-mercaptophenol (4-MPH) ligand. The surface-functionalized AuNRs, 4-MPH-AuNRs, were characterized by UV-Vis spectroscopy, dynamic light scattering (DLS), transmission electron microscopy (TEM) imaging, zeta potential, and Fourier-Transformed Infrared (FTIR) spectroscopy. The antibacterial and antibiofilm activities of 4-MPH-AuNRs were evaluated against a clinical isolate of Methicillin-Resistant Staphylococcus aureus (MRSA). The results indicate that the surface-modified nanorods, 4-MPH-AuNRs, exhibit a bactericidal activity with a minimum inhibitory concentration (MIC) of ~6.25 \(\mu\)g/ml against a planktonic suspension of MRSA. Furthermore, 4-MPH-AuNRs resulted in 2-3 log-cycle reduction of MRSA biofilm viable count over a concentration range of 100-4.0 \(\mu\)g/ml. The bacterial uptake of surface-modified nanorods was investigated by inductively coupled plasma-optical emission spectroscopy (ICP-OES) and scanning electron microscopy (SEM) imaging; the results reveal that ~27% of the nanorods were internalized into the bacterial cells after 6 hrs of exposure. SEM imaging revealed a significant accumulation of the nanorods at the bacterial cell wall and a possible cellular internalization. Thus, 4-MPH-AuNRs can be considered a potential novel antibacterial agent, particularly against resistant MRSA strain biofilm.

Ocurrence of Blandm-7 And Association With Blakpc-2, Blactx-M15, Aac, Aph, Mph(A), Catb3 And Virulence Genes In A Clinical Isolate of Klebsiella Pneumoniae With Different Plasmids In Brazil.

To characterize phenotypically and genotypically an isolate of Multidrug Resistant (MDR) K. pneumoniae from a patient with septicemia in a hospital in Recife-PE, Brazil, resistance and virulence genes were investigated by using PCR and sequencing the amplicons and the plasmid DNA was also sequenced. The K74-A3 isolate was resistant to all β-lactams, including carbapenems, as well as to aminoglycosides and quinolones. By conducting a PCR analysis and sequencing, the variant blaNDM-7 associated with blaKPC-2 and the cps, wabG, fimH, mrkD and entB virulence genes were identified. The analysis of plasmid revealed the presence of blaCTX-M15, aac(3)-IVa, aph(3')-Ia, aph(4)-Ia, aac(6')ib-cr, mph(A) and catB3, and also the plasmids IncX3, IncFIB, IncQ1, ColRNAI and ColpVC. To our knowledge, this is the first report of the blaNDM-7 gene in Brazil and we suggest that this variant is located in IncX3. These results alert us to the risk of spreading an isolate with a vast genetic arsenal of resistance, in addition to which several plasmids are present that favor the horizontal transfer of these genes.

Non-toxigenic Vibrio cholerae challenge strains for evaluating vaccine efficacy and inferring mechanisms of protection

Human challenge studies are instrumental for testing cholera vaccines, but these studies use outdated strains and require inpatient facilities. Here, we created next-generation isogenic Ogawa and Inaba V. cholerae challenge strains (ZChol strains) derived from a contemporary Zambian clinical isolate representative of current dominant pandemic V. cholerae. To minimize the risk of severe diarrhea these strains were rendered non-toxigenic, since antibody responses which limit V. cholerae colonization are the primary mechanism of immune protection. These strains did not cause diarrhea in infant mice and proved to accurately gauge reduction in intestinal colonization mediated by effective vaccination. They are also valuable as targets for measuring vibriocidal antibody responses. Using barcoded ZChol strains, we discovered that vaccination tightens the infection bottleneck without restricting pathogen expansion in vivo. ZChol strains have the potential to enhance the safety, relevance, and scope of future cholera vaccine challenge studies and be valuable reagents for studies of immunity to cholera.

Antibacterial effect of cerium oxide nanoparticle against Pseudomonas aeruginosa

Background Antibiotics have been widely used for the treatment of bacterial infections for decades. However, the rapid emergence of antibiotic-resistant bacteria has created many problems with a heavy burden for the medical community. Therefore, the use of nanoparticles as an alternative for antibacterial activity has been explored. In this context, metal nanoparticles have demonstrated broad-spectrum antimicrobial activity. This study investigated the antimicrobial activity of naked cerium oxide nanoparticles dispersed in aqueous solution (CNPs) and surface-stabilized using Pseudomonas aeruginosa as a bacterial model. Methods Gelatin-polycaprolactone nanofibers containing CNPs (Scaffold@CNPs) were synthesized, and their effect on P. aeruginosa was investigated. The minimum inhibitory and bactericidal concentrations of the nanoparticls were determined in an ATCC reference strain and a clinical isolate strain. To determine whether the exposure to the nanocomposites might change the expression of antibiotic resistance, the expression of the genes shv, kpc, and imp was also investigated. Moreover, the cytotoxicity of the CNPs was assessed on fibroblast using flow cytometry. Results Minimum bactericidal concentrations for the ATCC and the clinical isolate of 50 µg/mL and 200 µg/mL were measured, respectively, when the CNPs were used. In the case of the Scaffold@CNPs, the bactericidal effect was 50 µg/mL and 100 µg/mL for the ATCC and clinical isolate, respectively. Interestingly, the exposure to the Scaffold@CNPs significantly decreased the expression of the genes shv, kpc, and imp. Conclusions A concentration of CNPs and scaffold@CNPs higher than 50 μg/mL can be used to inhibit the growth of P. aeruginosa. The fact that the scaffold@CNPs significantly reduced the expression of resistance genes, it has the potential to be used for medical applications such as wound dressings.

Proteomic analysis of the colistin-resistant E. coli clinical isolate: Explorations of the resistome

Background: Antimicrobial resistance is a worldwide problem after the emergence of colistin resistance since it was the last option left to treat carbapenemase-resistant bacterial infections. The mcr gene and its variants are one of the causes for colistin resistance. Besides mcr genes, some other intrinsic genes are also involved in colistin resistance but still need to be explored. Objective: The aim of this study was to investigate differential proteins expression of colistin-resistant E. coli clinical isolate and to understand their interactive partners as future drug targets. Methods: In this study, we have employed the whole proteome analysis through LC-MS/MS. The advance proteomics tools were used to find differentially expressed proteins in the colistin-resistant Escherichia coli clinical isolate compared to susceptible isolate. Gene ontology and STRING were used for functional annotation and protein-protein interaction networks, respectively. Results: LC-MS/MS analysis showed overexpression of 47 proteins and underexpression of 74 proteins in colistin-resistant E. coli. These proteins belong to DNA replication, transcription and translational process; defense and stress related proteins; proteins of phosphoenol pyruvate phosphotransferase system (PTS) and sugar metabolism. Functional annotation and protein-protein interaction showed translational and cellular metabolic process, sugar metabolism and metabolite interconversion. Conclusion: We conclude that these protein targets and their pathways might be used to develop novel therapeutics against colistin-resistant infections. These proteins could unveil the mechanism of colistin resistance.

1230. Evaluation of the Interplay Between β-lactamases and Porin Production on β-Lactam MICs in Klebsiella pneumoniae

Background K. pneumoniae can emerge resistant to β-lactam antibiotics through the production of β-lactamase enzymes and/or loss of the outer membrane porins, OmpK35, OmpK36, and/or PhoE. While both mechanisms are hypothesized to work synergistically, β-lactamases have been the focus of previous studies. As a result, the contribution of outer membrane porin loss to the β-lactam minimum inhibitory concentration (MIC) is unknown. The objective of this study was to evaluate the contribution of specific β-lactamases and porin production to β-lactam susceptibility. We hypothesize that production of a β-lactamase in a clinical isolate deficient in 3 major porins will result in higher β-lactam MICs but not always a resistant phenotype. Methods The structural gene and promoter of CTX-M-14, CTX-M-15, and CMY-2 were cloned into a low copy number vector and transformed into Kp 23, a wild-type clinical isolate, and KPM 20, a clinical isolate deficient in OmpK35/36 and PhoE. MICs to ceftolozane/tazobactam, cefotaxime, ceftazidime, cefepime, and meropenem were determined by E-test. Kp 23 and KPM 20 were characterized by Western blot and whole genome sequencing. Results Production of CMY-2 alone led to a resistant phenotype for ceftolozane/tazobactam, cefotaxime, and ceftazidime regardless of porin production (Figure 1). CMY-2 production in KPM 20 resulted in non-susceptibility to meropenem. Both clones were susceptible to cefepime. Production of CTX-M-14 and CTX-M-15 in Kp 23 resulted in only cefotaxime resistance. Production of CTX-M-14 and CTX-M-15 in KPM 20 resulted in isolates non-susceptible to all antibiotics tested. Figure 1. MICs of K. pneumoniae clones against panel of β-lactam antibiotics. Conclusion When evaluating clinical isolates, it is impossible to determine the contribution of individual resistance mechanisms in the susceptibility pattern. This study demonstrated that resistance is not solely dependent on the β-lactamase produced and that the impact of porin deficiency varies with the antibiotic being evaluated. These data suggest that antibiotic selection may be more nuanced and that a broader range of therapeutics may be available given the appropriate diagnostic tools. Understanding the contributions of all resistance mechanisms is necessary to inform selection of the most appropriate antibiotic therapy. Disclosures Nancy D. Hanson, PhD, Merck (Grant/Research Support)

Whole-Genome Sequence of Streptococcus pyogenes Strain 591, Belonging to the Genotype emm 49

Streptococcus pyogenes strain 591 is a clinical isolate belonging to the genotype emm 49. It has been intensively studied for its pathogenicity traits. In this study, the complete genome of strain 591 was sequenced. It consists of a chromosome of 1,762,765 bp with a G+C content of 38.5%.

Cefazolin and Imipenem Enhance AmpC Expression and Resistance in NagZ-Dependent Manner in Enterobacter Cloacae

Background: Enterobacter cloacae (EC) is a commonly occurring opportunistic pathogen and is responsible for causing various infections in humans. Owing to its inducible chromosomal AmpC β-lactamase (AmpC), EC is inherently resistant to the 1st- and 2nd- generation cephalosporins. However, whether β-lactams antibiotics enhance EC resistance remains unclear.Results: In this study, we found that subinhibitory concentrations (SICs) of cefazolin (CFZ) and imipenem (IMP) are able to advance the expression of AmpC and improve its resistance towards β - lactams through NagZ in EC clinical isolate. Our work indicate that AmpC manifested a substantial upregulation in EC in response to SICs of CFZ and IMP. In nagZ knockout EC (ΔnagZ), we found that the resistance to β - lactam antibiotics was rather weakened and the effect of CFZ and IMP on induction of AmpC was completely abrogated. Ectopic expression of NagZ can rescue the induction effect of CFZ and IMP on AmpC and enhance resistance in ΔnagZ. More importantly, CFZ and IMP have the potential to bring about the target genes expressions of AmpR in a NagZ-dependent manner.Conclusions: Our findings show that NagZ is a critical determinant for CFZ and IMP to promote AmpC expression and improve resistance and that CFZ and IMP should be used with caution since they may aggravate EC resistance. At the same time, this study further improves our understanding of resistance mechanisms in EC.