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80 Acinetobacter strains were studied. Their differentiation according to the specific belonging showed that 55 strains belonged toA. calcoaceticusvar.anitratumspecies and 25 strains toA. calcoaceticusvar.lwoffi. It was established that Acinetobacter strains are multi dry resistant. In particular they showed 100% resistance to ampicillin, carbenicillin, erythromycin, methicillin, oxacillin, tetracycline, oleandomycin, streptomycin, rondomycin, linkomycin. According to the obtained data claphoran and phortum are the most effective.

Pathogenic bacteria of Scophthalmus maximus enteritis were isolated from and identified in an aquaculture farm in Changli County, Hebei Province, China. The feasibility of rapid selection of prevention and treatment drugs via PCR technology was investigated through drug resistance phenotype analysis and drug resistance gene detection. A dominant bacterial strain was isolated from S. maximus and designated HC-1. Morphological observations, culture property investigations, and physiological-biochemical experiments were conducted, which showed that the strain was consistent with the phenotypic characteristics of Vibrio. Based on 16S rRNA sequence determination and homology analysis, HC-1 was determined to be 99.65% similar to Vibrio harveyi NR 043165.1. Therefore, the bacterium was identified as V. harveyi. The sensitivity of the V. harveyi isolate to 41 antibiotics was determined by the Kirby-Bauer method. The results showed that HC-1 was resistant to 8 agents, namely, penicillin, oxacillin, amoxicillin, ampicillin, spectinomycin, azithromycin, florfenicol, and carbenicillin. The antimicrobial resistance gene analysis results showed that tem, ant(3˝)-I and tetA are carried by the chromosome of the strain and the plasmid, while sul1 is carried by the chromosome of the strain but not the plasmid. In contrast, floR is carried only by the plasmid but not by the chromosome. vanA, qnrA, and mefA are not carried by the plasmid or chromosome of the strain. Although the resistant phenotype of the strains is regulated by resistance genes, the relationship between the phenotype and genotype should be revealed by numerous databases. To accelerate the speed of treatment selection, it is necessary to apply PCR technology to detect resistance genes to replace drug sensitivity testing. These results provide useful information for further understanding the antimicrobial resistance factors and treatment of bacterial disease in S. maximus.

Background and Aim: Shewanella algae is ubiquitous in marine-associated environments and has been increasingly recognized as a significant human pathogen that can cause serious infections mainly associated with exposure to seawater and ingestion of raw seafood. This study aimed to isolate and characterize S. algae from ballast water of ships berthed at Port Klang, Malaysia. Materials and Methods: Ballast water was sampled from nine ships docked at Port Klang, Malaysia. The isolates were identified and characterized based on biochemical and enzymatic properties, 16S rRNA and gyrB sequencing, biofilm formation capability, and antibiotic susceptibility. Results: A total of four S. algae isolates were isolated from four ballast water samples tentatively name Sa-BW1, Sa-BW2, Sa-BW7, and Sa-BW8. All isolates showed positive reaction for cytochrome oxidase, catalase, high tolerance to NaCl (6% and 8%), ability to grow at 42°C, and on Salmonella-Shigella agar. The strains also exhibited β-hemolytic activity on sheep blood and human blood agar, positive reaction for lipase, protease, DNase and gelatinase, strong biofilm adherence capabilities and multiple antibiotic resistances against ampicillin, carbenicillin, cephalothin, colistin, novobiocin, oxacillin, penicillin, rifampicin, and tobramycin which suggested their potential pathogenicity. Conclusion: This study demonstrated the occurrence of putative pathogen S. algae in ballast water of ships docked at Malaysian port.

Burn wound infections with multidrug-resistant (MDR) bacteria are shown in many countries as severe widespread health threats. Consequently, attention has been devoted to new nanoparticle-based materials in the field of antimicrobial chemotherapy for burn wound infections. This study aimed to evaluate both in vitro and in vivo efficacies of nanoparticle–antibiotic combinations as new classes of materials subjected against MDR Pseudomonas aeruginosa. Out of 40 Gram-negative isolates, 23 P. aeruginosa were recovered from patients with burn wound infections attending different hospitals in Tanta, Egypt. The susceptibility test revealed that 95.7% of P. aeruginosa isolates were MDR with a high incidence of resistance against carbenicillin. Antibacterial activities of silver nanoparticles (Ag-NPs) against the isolates examined showed various inhibition zone diameters ranging from 11 to 17 mm. Strong synergistic efficacy of neomycin was reported in combination with Ag-NPs against MDR P. aeruginosa P8 and P14 isolates. The in vivo effectiveness of various pharmaceutical formulations prepared from a combination of neomycin antibiotic with Ag-NPs in the treatment of induced bacterially infected mice burns showed that maximum healing activity along with faster wound contraction reported with the combination of neomycin-Ag-NPs in the spray formulation. Generally, data indicated that incorporating Ag-NPs in combination with certain antibiotics may be a new, promising application for wound treatments, especially burns infected with MDR P. aeruginosa.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that presents a serious risk to immunosuppressed individuals and other extremely vulnerable patients such as those in intensive care units. The emergence of multidrug-resistant Pseudomonas strains has increased the need for new antipseudomonal agents. In this study, a series of amino group-modified aminopenicillin derivatives was synthesized that have different numbers of carboxyl groups and structurally resemble carboxypenicillin-ureidopenicillin hybrids, and their antipseudomonal activities were evaluated. Among the derivatives synthesized, diethylenetriaminepentaacetic acid (DTPA)-modified amoxicillin (DTPA-Amox) showed potent antipseudomonal activity, not only against the laboratory strain PAO1 but also against clinically isolated Pseudomonas strains that were resistant to piperacillin and carbenicillin. DTPA-Amox had no obvious cytotoxic effects on cultured mammalian cells. In addition, in an in vivo model of leukopenia, DTPA-Amox treatment produced a moderate but statistically significant improvement in the survival of mice with P. aeruginosa strain PAO1 infection. These data suggest that polycarboxylation by DTPA conjugation is an effective approach to enhance antipseudomonal activity of aminopenicillins.

The emergence of methicillin-resistant Staphylococcus aureus (MRSA) infection is one of the greatest threats to both animal and human health. Our investigation was aimed to identify and differentiate between MRSA and methicillin-sensitive Staphylococcus aureus (MSSA) recovered from mastitic milk using MALDI-TOF mass spectrometry compared with phenotypic methods and studying their susceptibility to various antibiotics. Four hundred milk samples from mastitic animals (cows, sheep, goats, and dromedary camels) were investigated. Phenotypic identification of S. aureus was made through MASTASAPH Latex test, STAPH ID 32, and Vitek 2 system. The proteomic characterization of S. aureus was done by MBT. The Kirby Bauer method was accomplished to detect the resistance of S. aureus strains to antibiotics. The results of the MASTASAPH Latex test, revealed that 54 (46%) were recognized as S. aureus. All S. aureus isolates were identified by MBT with a score of more or equal 2.00. Several peaks were identified in the mass of 4590 Da, 4863 Da, and 4938 Da for MSSA and in the mass of 2636 Da and 3009 Da for MRSA. The MSP dendrogram demonstrated that the S. aureus isolates were classified into one group with a distance level of less or equal 400. The percentage of S. aureus resistance against carbenicillin, erythromycin and kanamycin was 94.4%, 38.88%, and 33.33%, respectively. In conclusion, S. aureus bacteria are among the key triggers for mastitis in Saudi Arabia. MBT is reported to be not only the rapid tool to identify S. aureus but also able to discriminate MRSA from MSSA.

Probiotics serving as an alternative to the criticized antibiotics mainly focus on improving animal's growth and health. After realizing the dangers posed by diseases that have led to lots of economic losses, aquaculture scientists have sought the usage of probiotics. However, most probiotics are ineffective in eliciting aquatic animals' preferred effects, since they are from non-fish sources. Again, there are even a few marine aquatic probiotics. Given this, a study was conducted to investigate the probiotic potential of the bacteria species isolated from the digestive tract of hybrid grouper (Epinephelus fuscoguttatus♀ × Epinephelus lanceolatus♂). Based on the morphological, biochemical, 16S rRNA sequencing analysis and evolutionary relationships, the isolated species were identified as Bacillus tequilensis GPSAK2 (MW548630), Bacillus velezensis GPSAK4 (MW548635), and Bacillus subtilis GPSAK9 (MW548634), which were designated as GPSAK2, GPSAK4, and GPSAK9 strains, respectively. Their probiotic potentials including their ability to tolerate high bile salt concentration, low pH, high temperatures, adhesion ability (auto-aggregation and cell-surface hydrophobicity), antimicrobial activity and biosafety test, compatibility test, hemolytic activity, and antibiotic susceptibility test were evaluated. While GPSAK2 and GPSAK9 strains were γ-hemolytic, that of GPSAK4 was α-hemolytic. All the isolates were resistant to low pH (1) and higher bile salt concentration (0.5%), showed higher viability ability after higher temperature exposure (80, 90, and 100°C), as well as higher cell-surface percentage hydrophobicity and auto-aggregation. All isolates exhibited positive compatibility with each other, signifying their ability to be used as multispecies. The three strains were susceptible to ampicillin (except GPSAK9, which was resistant), penicillin, kanamycin, ceftriaxone, chloramphenicol, erythromycin, clindamycin, furazolidone (except GPSAK2 and GPSAK9, which were moderately susceptible and resistant, respectively), polymyxin B, vancomycin (except GPSAK9, which was resistant), sulfamethoxazole (except GPSAK9, which was moderately susceptible), amikacin, minocycline, ofloxacin, norfloxacin, doxycycline, neomycin, gentamicin, tetracycline, carbenicillin, midecamycin (except GPSAK9, which was moderately susceptible), ciprofloxacin, piperacillin, and cefoperazone. All isolates demonstrated good antimicrobial activity against four pathogens, viz. Streptococcus agalactiae, Streptococcus iniae, Vibrio harveyi, and Vibrio alginolyticus. The results collectively suggest that Bacillus strains GPSAK2, GPSAK4, and GPSAK9 could serve as potential probiotic candidates that can be used to improve the growth and health status of aquatic animals, especially grouper.

The phenotypic characteristics and growth kinetics at several temperatures, salinities, and pH values of three Vibrio parahaemolyticus (Vp) strains with different virulence and one nonpathogenic strain were evaluated. Independent of the virulence of the strain, a high metabolic diversity was found, which yielded different colored phenotypes on the CHROMagar™ Vibrio. All strains were resistant to ampicillin and carbenicillin, and Vp AHPND+ organisms were the most sensitive to enrofloxacin. The exponential growth of Vp strains started at 1–2 hr of incubation, although no relationship was observed between the bacterial density and degree of virulence. Moreover, the growth of the most virulent strain was independent of the nutrients in the incubation media during the initial hour postinoculation. No strain grew at 4°C in 0% NaCl and pH 4, but only Vp AHPND+ grew at 44°C. For all strains, the lag phase was proportional to the NaCl concentration, and the growth was better at pH 8–9. However, the Vp AHPND− strain displayed a greater variability, was more sensitive to extreme conditions, and showed a lag phase of 9 hr independent of the pH.

Current study aimed to analyze the synergistic killing of pathogenic Escherichia coli using camel lactoferrin from different Saudi camel clans and various antibiotics. Methods: using multiple microbiological and protein analysis techniques, the results were shown that the purified camel lactoferrins (cLfs) from different Saudi camel have strong antimicrobial potentials against two strains of E. coli. Although all cLfs were superior relative to human or bovine lactoferrins (hLf or bLf), there was no noticeable difference in the antimicrobial potentials of cLfs from different camel clans. The effects of antibiotics and cLfs were synergistic, indicating the superiority of using cLf-antibiotic combinations against E. coli growth. Since these combinations possessed distinguished synergy profiles, it is likely that they can be used to enhance the low efficacy of antibiotics, as well as to control the problems associated with bacterial resistance. Furthermore, these combinations can reduce the cost of cure of bacterial infections, especially in the developing countries. The analysis of the molecular mechanisms of lactoferrin action revealed that expression of several E. coli proteins was affected by the treatment with these antibacterial factors. Several proteins of different molecular weights interacting with cLf-biotin were found. Scanning and transmission electron microscopy analysis revealed the presence of noticeable morphological changes associated with the treatment of E. coli strains by antibiotic carbenicillin or cLf alone, and in combination. Camel lactoferrin has superior potential killing of E. coli over bovine and human lactoferrin, and this potential can be further synergistically enhanced of cLF is combined with antibiotics.

The emergence and spread of extended-spectrum β-lactamases (ESBLs), metallo-β-lactamases (MBLs), or variant low-affinity penicillin-binding proteins (PBPs) pose a major threat to our ability to treat bacterial infection using β-lactam antibiotics. Although combinations of β-lactamase inhibitors with β-lactam agents have been clinically successful, there are no MBL inhibitors in current therapeutic use. Furthermore, recent clinical use of new-generation cephalosporins targeting PBP2a, an altered PBP, has led to the emergence of resistance to these antimicrobial agents. Previous work shows that natural polyphenols such as cranberry-extracted proanthocyanidins (cPAC) can potentiate non-β-lactam antibiotics against Gram-negative bacteria. This study extends beyond previous work by investigating the in vitro effect of cPAC in overcoming ESBL-, MBL-, and PBP2a-mediated β-lactam resistance. The results show that cPAC exhibit variable potentiation of different β-lactams against β-lactam-resistant Enterobacteriaceae clinical isolates as well as ESBL- and MBL-producing E. coli. We also discovered that cPAC have broad-spectrum inhibitory properties in vitro on the activity of different classes of β-lactamases, including CTX-M3 ESBL and IMP-1 MBL. Furthermore, we observe that cPAC selectively potentiate oxacillin and carbenicillin against methicillin-resistant but not methicillin-sensitive staphylococci, suggesting that cPAC also interfere with PBP2a-mediated resistance. This study motivates the need for future work to identify the most bioactive compounds in cPAC and to evaluate their antibiotic-potentiating efficacy in vivo. IMPORTANCE The emergence of β-lactam-resistant Enterobacteriaceae and staphylococci compromises the effectiveness of β-lactam-based therapy. By acquisition of ESBLs, MBLs, or PBPs, it is highly likely that bacteria may become completely resistant to the most effective β-lactam agents in the near future. In this study, we described a natural extract rich in proanthocyanidins which exerts adjuvant properties by interfering with two different resistance mechanisms. By their broad-spectrum inhibitory ability, cranberry-extracted proanthocyanidins could have the potential to enhance the effectiveness of existing β-lactam agents.

Some Vibrio species are important human pathogens owing to they cause infectious diseases such as gastroenteritis, wound infections, septicemia or even death. Many of these illnesses are associated with consumption of contaminated seafood. In the present study, we evaluated the presence of pathogenic Vibrio species, their virulence and antimicrobial susceptibility from 285 different kind of seafood samples from "La Nueva Viga" market in Mexico City. The PCR assay was used for amplification the vppC (collagenase), vmh (hemolysin), tlh (thermolabile hemolysin), and vvhA (hemolytic cytolysin) genes that are specific to Vibrio alginolyticus (detected in 27%), Vibrio mimicus (23.2%), Vibrio parahaemolyticus (28.8%) and Vibrio vulnificus (21.1%), respectively. Several genes encoding virulence factors were amplified. These included V. alginolyticus: pvuA (17.9%), pvsA (50%), wza and lafA (100%); V. mimicus: iut A (60%), toxR (100%); V. parahaemolyticus: pvuA (58.7%), pvsA (26.1%), wza (2.2%), and lafA (100%); and V. vulnificus: wcrA (77.5%), gmhD (57.5%), lafA (100%) and motA (30%). The antibiotic susceptibility of the Vibrio species isolates revealed that most of them were resistant to ampicillin, cephalothin and carbenicillin but susceptible to pefloxacin and trimethoprim-sulfamethoxazole. Our results indicated a high prevalence of pathogenic Vibrio species in seafood, a high presence of virulence genes and that Vibrio species continuously exposed to antibiotics, therefore, consumption of these kind of seafood carries a potential risk for foodborne illness.

The remarkable ability of Agrobacterium tumefaciens to transfer DNA to plant cells has allowed the generation of important transgenic crops. One challenge of A. tumefaciens-mediated transformation is eliminating the bacteria after plant transformation to prevent detrimental effects to plants and the release of engineered bacteria to the environment. Here we use a reverse genetics approach to identify genes involved in ampicillin resistance with the goal of utilizing these antibiotic-sensitive strains for plant transformations. We show that treating A. tumefaciens C58 with ampicillin led to increased β-lactamase production, a response dependent on the broad-spectrum β-lactamase AmpC and its transcription factor AmpR. Loss of the putative ampD orthologue, atu2113, led to constitutive production of AmpC-dependent β-lactamase activity and ampicillin resistance. Finally, one cell wall remodeling enzyme, MltB3, was necessary for the AmpC-dependent β-lactamase activity and its loss elicited ampicillin and carbenicillin sensitivity in the A. tumefaciens C58 and GV3101 strains. Furthermore, GV3101 ΔmltB3 transforms plants with comparable efficiency to wildtype but can be cleared with sub-lethal concentrations of ampicillin. The functional characterization of the genes involved in the inducible ampicillin resistance pathway of A. tumefaciens constitutes a major step forward in efforts to reduce the intrinsic antibiotic resistance of this bacterium.IMPORTANCEAgrobacterium tumefaciens, a significant biotechnological tool for production of transgenic plant lines, is highly resistant to a wide variety of antibiotics, posing challenges for various applications. One challenge is the efficient elimination of A. tumefaciens from transformed plant tissue without using levels of antibiotics that are toxic to the plants. Here, we present the functional characterization of genes involved in β-lactam resistance in A. tumefaciens. Knowledge about proteins that promote or inhibit β-lactam resistance will enable the development of strains to improve the efficiency of Agrobacterium-mediated plant genetic transformations. Effective removal of Agrobacterium from transformed plant tissue has the potential to maximize crop yield and food production, improving the outlook for global food security.

Transpeptidation reinforces the structure of cell-wall peptidoglycan, an extracellular heteropolymer that protects bacteria from osmotic lysis. The clinical success of transpeptidase-inhibiting β-lactam antibiotics illustrates the essentiality of these cross-linkages for cell-wall integrity, but the presence of multiple, seemingly redundant transpeptidases in many species makes it challenging to determine cross-link function. Here, we present a technique to link peptide strands by chemical rather than enzymatic reaction. We employ biocompatible click chemistry to induce triazole formation between azido- and alkynyl-d-alanine residues that are metabolically installed in the peptidoglycan of Gram-positive or Gram-negative bacteria. Synthetic triazole cross-links can be visualized using azidocoumarin-d-alanine, an amino acid derivative that undergoes fluorescent enhancement upon reaction with terminal alkynes. Cell-wall stapling protects Escherichia coli from treatment with the broad-spectrum β-lactams ampicillin and carbenicillin. Chemical control of cell-wall structure in live bacteria can provide functional insights that are orthogonal to those obtained by genetics.

Silver nanoparticles (AgNPs) are promising alternatives to antibiotics. The aims of this study were to produce AgNPs using two biological methods and determine their antibacterial activity against Pseudomonas aeruginosa and Staphylococcus pseudintermedius. AgNPs were biosynthesized from an infusion of Curcuma longa (turmeric) and the culture supernatant of E. coli. Characterization was achieved by ultraviolet-visible spectroscopy and by Transmission Electron Microscopy (TEM). The antibacterial properties of NPs from C. longa (ClAgNPs) and E. coli (EcAgNPs), alone and in combination with carbenicillin and ampicillin, were investigated through the Kirby-Bauer disk diffusion assay and the minimum inhibitory concentration (MIC). Dimensions of NPs ranged from 11.107 &plusmn, 2.705 nm (ClAgNPs) to 27.282 &plusmn, 2.68 nm (EcAgNPs). Kirby-Bauer and MIC assays showed great antibacterial abilities for both NPs alone and in combination with antibiotics. EcAgNPs alone showed the most powerful antibacterial activities, resulting in MIC values ranging from 0.438 &plusmn, 0.18 &micro, M (P. aeruginosa) to 3.75 &plusmn, 3.65 &micro, M (S. pseudintermedius) compared to those of ClAgNPs: 71.8 &plusmn, 0 &micro, M (P. aeruginosa) and 143.7 &plusmn, M (S. pseudintermedius). The antibiofilm abilities were strain-dependent, but no statistical differences were found between the two NPs. These results suggest the antibacterial potential of AgNPs for the treatment of infectious diseases.

[EN] The presence of antibiotics in soils could be due to natural production by soil microorganisms or to the effect of anthropogenic activities. However, the impact of these compounds on plant physiology has not been thoroughly investigated. To evaluate the effect of beta-lactam antibiotics (carbenicillin and penicillin) on the growth and development of Arabidopsis thaliana roots, plants were grown in the presence of different amounts and we found a reduction in root size, an increase in the size of root hairs as well as an abnormal position closer to the tip of the roots. Those phenomena were dependent on the accumulation of both antibiotics inside root tissues and also correlated with a decrease in size of the root apical meristem not related to an alteration in cell division but to a decrease in cell expansion. Using an RNA sequencing analysis, we detected an increase in the expression of genes related to the response to oxidative stress, which would explain the increase in the levels of endogenous reactive oxygen species found in the presence of those antibiotics. Moreover, some auxin-responsive genes were misregulated, especially an induction of CYP79B3, possibly explaining the increase in auxin levels in the presence of carbenicillin and the decrease in the amount of indole glucosinolates, involved in the control of fungal infections. Accordingly, penicillin-treated plants were hypersensitive to the endophyte fungus Colletotrichum tofieldiae. These results underscore the risks for plant growth of beta-lactam antibiotics in agricultural soils, and suggest a possible function for these compounds as fungus-produced signaling molecules to modify plant behavior., This work has been financed by the Spanish Ministerio de Medio Ambiente Rural y Marino [MMA 022/PC08/3-04.2] and the Spanish Agencia Estatal de Investigación [BFU2016-80621-P]. M.E.G. is a fellow of the program Convocatoria Abierta 2013 - Fase I (Instituto de Fomento de Talento Humano (IFTH), Secretaría Nacional de Educación Superior Ciencia y Tecnología (SENESCYT) (Ecuador). N.B-T is a recipient of a MINECO FPI Fellowship (BES-2014-068868).

There is a general lack of understanding about how communities of bacteria respond to exogenous toxins such as antibiotics. Most of our understanding of community-level stress responses comes from the study of stationary biofilm communities. Although several community behaviors and production of specific biomolecules affecting biofilm development and associated behavior have been described for Pseudomonas aeruginosa and other bacteria, we have little appreciation for the production and dispersal of secreted metabolites within the 2D and 3D spaces they occupy as they colonize, spread, and grow on surfaces. Here we specifically studied the phenotypic responses and spatial variability of alkyl quinolones, including the Pseudomonas quinolone signal (PQS) and members of the alkyl hydroxyquinoline (AQNO) subclass, in P. aeruginosa plate-assay swarming communities. We found that PQS production was not a universal signaling response to antibiotics, as tobramycin elicited an alkyl quinolone response, whereas carbenicillin did not. We also found that PQS and AQNO profiles in response to tobramycin were markedly distinct and influenced these swarms on different spatial scales. At some tobramycin exposures, P. aeruginosa swarms produced alkyl quinolones in the range of 150 μm PQS and 400 μm AQNO that accumulated as aggregates. Our collective findings show that the distribution of alkyl quinolones can vary by several orders of magnitude within the same swarming community. More notably, our results suggest that multiple intercellular signals acting on different spatial scales can be triggered by one common cue.

Aeromonads represent bacteria thought to be primarily mostly autochthonous to aquatic environments. This study was focused on the relation with antibiotics and enterocins of identified Aeromonas species isolated from the intestine of trouts living in Slovakian aquatic sources. Intestinal samples from 50 trouts (3 Salmo trutta and 47 Salmo gairdnerii) were collected in April of years 2007, 2010, and 2015 from trouts of different water sources in Slovakia (pond Bukovec near Košice, river Čierny Váh). Due to the MALDI-TOF mass spectrometry evaluation, 25 strains were proposed to the genus Aeromonas involving nine different species (Aeromonas bestiarum-nine strains, Aer. salmonicida-four strains, Aer. encheleia, Aer. eucrenophila, Aer. molluscorum, Aer. media, Aer. sobria, Aer. popoffii, Aer. veronii). Phenotypic evaluation of individual strains confirmed their species identification. Twenty-five strains of different Aeromonas species were sensitive to azithromycin, amikacin, mecillinam, mezlocillin, piperacillin, gentamicin, chloramphenicol, and tetracycline. On the other side, they were resistant to carbenicillin and ticarcillin. The growth of Aer. bestiarum R41/1 was inhibited by treatment with Ent M and Ent 2019 (inhibition activity 100 AU/mL). Aer. bestiarum R47/3 was inhibited by eight enterocins (100 AU/mL). It is the first study testing enterocins to inhibit the growth of Aeromonas species from trouts.

Drug-resistant Salmonella is frequently detected in most parts of the world, and its rate of resistance has increased significantly in recent years. However, this study aimed to evaluate the minimum inhibitory concentration (MIC, determined with the Kirby-Bauer method) of quinolones in 86 Salmonella spp. strains isolated from pigs. Both the inside and outside of the QRDR region of strains were sequenced. The DNA sequence of the QRDR region of Salmonella spp. revealed the mutations S83F, D87N and S83Y. The region outside the QRDR showed a mutation in L582G. Forty-five isolates of Salmonella ssp. were categorized as quinolone-resistant; out of these, 16 corresponded to Salmonella enterica and isolates showed intermediate resistance (6.25%) to nalidixic acid. Three isolats (18.6%) were resistant to ampicillin; two (12.5%) were resistant to carbenicillin. Moreover, three (18.7%) isolates were resistant to gentamicin, nitrofurantoin and pefloxacin, and 8 (50%) were resistant to trimethoprim/sulfamethoxazole. Six percent of Salmonella spp. strains showed less resistance to antimicrobial agents compared to S. Thyphimurium (18%). The resistance to individual quinolones varied by serotypes. For S. anatum and S. Reading, it was 12.25%, and for S. choreaeaesuis, S. typhimurium monofasica, 6.25%. In contrast, S. agona, S. bredeney and S. london were sensitive to these antibiotics. In conclusion, quinolones have become the drugs of choice for the treatment of severe Salmonella infections. The study of mutations outside the QRDR region opens up new insights about the resistance of Salmonella to fluoroquinolones.

Summary Nucleic acid amplification‐based methods are limited by their inability to discriminate between viable and dead cells. To overcome this drawback, propidium monoazide (PMA) combined with qPCR has been used to differentiate viable from nonviable cells in environmental samples. However, assessing bacterial physiology using PMA‐qPCR remains a challenge due to its incapability of detecting metabolic activities, leading to overestimation of the viable bacteria population under an inactivation condition (e.g. antibiotic treatments). A recent advanced technique to amplify ribosomal RNA precursors (pre‐rRNA) has been shown to detect viable cells because pre‐rRNAs are intermediates in rRNA synthesis. This study investigated the effect of different types of antibiotics on the bacterial viability or viable but non‐culturable (VBNC) state using both PMA‐qPCR and pre‐rRNA analyses with Pseudomonas aeruginosa. This study demonstrated that P. aeruginosa was more sensitive to colistin than it was to carbenicillin, gentamicin and levofloxacin. We could discriminate VBNC P. aeruginosa cells using PMA‐qPCR when antibiotic pressure induced the VBNC state. Also, pre‐rRNA was able to distinguish viable cells from colistin‐inactivated bacteria cells, and it could detect the presence of VBNC and persister cells. Our results showed that these two molecular methods could successfully eliminate false‐positive signals derived from antibiotics‐inactivated cells.

Isolated Agrobacterium tumefaciens was exposed to different extremely low frequencies of square amplitude modulated waves (QAMW) from two generators to determine the resonance frequency that causes growth inhibition. The carrier was 10 MHz sine wave with amplitude ±10 Vpp which was modulated by a second wave generator with a modulation depth of ± 2Vpp and constant field strength of 200 V/m at 28 °C. The exposure of A. tumefaciens to 1.0 Hz QAMW for 90 min inhibited the bacterial growth by 49.2%. In addition, the tested antibiotics became more effective against A. tumefaciens after the exposure. Furthermore, results of DNA, dielectric relaxation and TEM showed highly significant molecular and morphological changes due to the exposure to 1.0 Hz QAMW for 90 min. An in-vivo study has been carried out on healthy tomato plants to test the pathogenicity of A. tumefaciens before and after the exposure to QAMW at the inhibiting frequency. Symptoms of crown gall and all pathological symptoms were more aggressive in tomato plants treated with non-exposed bacteria, comparing with those treated with exposed bacteria. We concluded that, the exposure of A. tumefaciens to 1.0 Hz QAMW for 90 min modified its cellular activity and DNA structure, which inhibited the growth and affected the microbe pathogenicity.

Resumen Introducción: Los alimentos de origen animal frecuentemente están implicados en brotes de salmonelosis. Objetivo: Evaluar la frecuencia de Salmonella enterica en carnes molidas de pollo, res y cerdo (un total de 2.592 muestras) obtenidas de mercados sobre ruedas y supermercados de la Delegación Iztapalapa en la Ciudad de México, determinar la susceptibilidad antimicrobiana y efectuar ensayos de adherencia en las cepas aisladas. Métodos: El aislamiento de S. enterica se hizo de acuerdo a la BAM-FDA, la susceptibilidad antimicrobiana de acuerdo con CLSI y el ensayo de adherencia en células HEp-2 conforme a Baffone y cols., 2001. Resultados: Salmonella enterica fue aislada en 511 del total de muestras analizadas (19,7%), de las cuales 244 (47,7%), 152 (29,7%) y 115 (22,5%) correspondieron a carne molida de pollo, res y cerdo, respectivamente. La mayor frecuencia de resistencia de S. enterica a antimicrobianos fue a ampicilina y cloranfenicol en pollo, perfloxacina y ampicilina en res y carbenicilina, ampicilina, cloranfenicol, cefotaxima y perfloxacina en cerdo. Noventa por ciento de las cepas mostraron un patrón de adherencia agregativo. Conclusión: La frecuencia de S. enterica en productos cárnicos es alta, por lo que es importante la adecuada cocción de la carne para disminuir el riesgo de una salmonelosis.

The goal of the work was to analyze the ability of natural and semisynthetic penicillins to regulate the quorum sensing system in bacteria. The test strain was C. violaceum NCTC 13274 (CV 026) bearing mini-Tn5 insertion in the cviI gene that led to irreversible loss of N-acyl-L-homoserine lactone (AHL) biosynthesis and reversible suppression of violacein biosynthesis. Within the temperature range suboptimal for C. violaceum growth (17–22°C), subinhibitory concentrations of piperacillin, azlocillin, ticarcillin, carbenicillin, and oxacillin showed a quorum-dependent induction of violacein production, probably acting as AHL-mimetics. Cleavage of these antibiotics with beta-lactamase I and II led to the loss of both antibacterial and quorum-inducing activities. Inhibition of C. violaceum beta-lactamase by sulbactam resulted in manifestation of combined antibacterial and quorum-inducing activity of ampicillin and amoxicillin. These results improve our understanding of the role of beta-lactams and beta-lactamases in the processes of interspecific antagonism and in microbial intercellular communication.

The widespread bacterial resistance to a broad range of antibiotics necessitates rapid antibiotic susceptibility testing before effective treatment could start in the clinic. Among resistant bacteria, Staphylococcus aureus is one of the most important, and Methicillin-resistant (MRSA) strains are a common cause of life threatening infections. However, standard susceptibility testing for S. aureus is time consuming and thus the start of effective antibiotic treatment is often delayed. To circumvent the limitations of current susceptibility testing systems, we designed an assay that enables measurements of bacterial growth with higher spatial and temporal resolution than standard techniques. The assay consists of arrays of microwells that confine small number of bacteria in small spaces, where their growth is monitored with high precision. These devices enabled us to investigate the effect of different antibiotics on S. aureus growth. We measured the Minimal Inhibitory Concentration (MIC) in less than 3 hours. In addition to being significantly faster than the 48 hours needed for traditional microbiological methods, the assay is also capable of differentiating the specific effects of different antibiotic classes on S. aureus growth. Overall, this assay has the potential to become a rapid, sensitive, and robust tool for use in hospitals and laboratories to assess antibiotic sensitivity.

Microbial lipases today occupy a place of prominence among biocatalysts owing to their ability to catalyze awide variety of reactions in aqueous and non- aqueous media, A.baumannii were isolated from different clinical specimens from hospitalized patients from Baghdad hospitals and were detected by biochemical tests and API20E system. The percentage of isolation was (16.6%), A. baumannii is an increasingly multidrug – resistant (MDR), it showed high level of resistant to Ceftriaxon, Colistin, Piperacillin, Co-trimoxazol, Tertracycline, Carbenicillin, Amoxicillin, Penicillin G, Gentamicin and Ceftazidim , wherease the isolates were highly sensitive to Imipenem, Ciprofloxacin, Meropenem, Amikacin, and Cefotaxime. The isolated strians of A.baumannii were screened for the production of lipase by using Rhodamine B agar media. one of the isolated strians exhibited a greater clear zone than the others, indicated higher lipase activity. The lipase in present study was partially purified by ammonium sulphate at the concenterations of 30% and 80%, the 30% ammonium sulphate showed higher activiy of the enzyme than 80% in which the enzyme activity was slightly decreased indicating that the better purification was at the 30% concenteration of ammonium sulphate.Various physicochemical parameters such as pH, temperature and metal ions were studied in order to determine the optimum conditions for lipase production. The production of lipase by A. baumannii was optimum at 35°C, pH 7.0 and was enhanced by the Ca++ and Na+ wherease inhibited by Zn++ ions. Key words : Acinetobacter baumannii, Lipase enzyme, Partial purification.

Background Infections caused by Acinetobacter baumannii were responsive to conventional antibiotic therapy. However, recently, carbapenem-associated multidrug resistant isolates have been reported worldwide and present a major therapeutic challenge. Epigallocatechin-3-Gallate (EGCG) extracted from green tea exhibits antibacterial activity. Purpose We evaluated the antibacterial activity of EGCG and possible synergism with antibiotics in carbapenem-associated multidrug resistant A. baumannii. A potential mechanism for synergism was also explored. Materials and methods Seventy clinical isolates of A. baumannii collected from geographically different areas were analyzed by minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of EGCG. Checkerboard and time-killing assays were performed to exam the synergism between EGCG and antibiotics. The effects of EGCG on a multidrug efflux pump inhibitor (1-[1-naphthylmethyl] piperazine; NMP) and β-lactamase production were also examined in A. baumannii. Results Sixty-three of 70 clinical isolates of A. baumannii carried carbapenemase-encoding genes with carbapenem-associated multidrug resistance. Levels of MIC and MBC of EGCG ranged from 64 to 512 µg/ml and from 128 to ≥ 1024 µg/ml, respectively among the clinical isolates. MIC90 and MBC86 levels were 256 µg/ml and 512 µg/ml of EGCG, respectively. Subinhibitory concentration of EGCG in combination with all antibiotics tested, including carbapenem, sensitized (MICs fall ≤ 1.0 µg/ml) all carbapenem-associated multidrug resistant isolates. Checkerboard and time-killing assays showed synergism between EGCG and meropenem (or carbenicillin) counted as fractional inhibitory concentration of 2log10 within 12 h, respectively. EGCG significantly increased the effect of NMP but was unrelated to β-lactamase production in A. baumannii, suggesting EGCG may be associated with inhibition of efflux pumps. Conclusion Overall we suggest that EGCG-antibiotic combinations might provide an alternative approach to treat infections with A. baumannii regardless of antibiotic resistance.

Rationale: Most cases of listeriosis are caused by the pathogenic Listeria monocytogenes. Some cases of isolation of L. innocua with pathogenicity factors from foods have been published, as well as on the cases of the disease in humans caused by this species. Aim: To assess biological properties including potential pathogenicity of L. innocua, isolated from food and environmental objects. Materials and methods: We performed microbiological study of L. innocua cultures isolated from foods (n = 35) and environmental objects (n = 15) on the territory of Primorye Territory (Russian Federation), as well as assessment of their sensitivity to antibiotics. Results: The studied L. innocua cultures showed stable phenotypic features of their biological properties, such as morphology, typical colony growth on the medium with characteristic odor of fermented milk, blue or blue-green luminescence induced by inclined light, presence of catalase activity and absence of the oxidase activity. Only 38 ± 6.9% of L. innocua demonstrated movements at T 22 °С. L. innocua cultures did not ferment mannitol (100% of cultures); they degraded ramnose to its acid without gas (70 ± 6.5%) and degraded xylose (42.8 ± 7%). Listeria isolated from vegetables and environmental objects could ferment ramnose (92.8 ± 7.2% of the studied cultures) and xylose (28.5 ± 12.5%) more frequently than L. innocua isolated from meat and fish foods. L. innocua demonstrated variable biochemical activities towards mannose (92 ± 3.8%), saccharose (85.7 ± 7.8%) and melesitose (76.2 ± 9.5%). L. innocua cultures with hemolytic activity (34 ± 6.7%) (α or β type) were isolated, more commonly from fish products. All Listeria irrespective of their isolation source showed lipase activity. L. innocua cultures from foods and environmental objects were highly sensitive to antimicrobials from the following classes: penicillins (ampicillin, carbenicillin, combined amoxicillin and clavulanic acid), aminoglycosides (gentamycin, amikacin), carbapenems (meropenem), and fluoroquinolones (ofloxacin). Conclusion: Some biological properties of L. innocua were variable depending on the source of isolation. Isolation of Listeria with atypical properties in theterritory of Primorye Territory requires that these microorganisms should be studied in more detail.

β-Lactams are the most widely used antibiotics in treating bacterial infections. However, they are rarely applied in infections caused by Vibrio parahaemolyticus, as the bacterium is intrinsically resistant to penicillins by expressing β-lactamase. Here we report structural characterization of the CARB β-lactamase from V. parahaemolyticus (CARB-20). CARB-20 is a class A β-lactamase, belonging to subclass A1 (containing 70STFKAL75, 130SDNTAANL137, 164RXEXXLN170, 231VGDKTG236, etc.), group LSBL2 (with the disulfide bridge C77–C123, motif 231IADRSGAG238 and R244). CARB-20 adopts a typical subclass A1 β-lactamase fold consisting of two domains. Its active site is constituted by four conserved motifs, similar to that of known subclass A1 β-lactamases. Analysis of the active site structure reveals its substrate preference for penicillin, ampicillin and carbenicillin but not for latterly developed cephalosporins. Meanwhile, β-lactamase inhibitors such as clavulanate and sulbactam can well fit into the active site, supporting β-lactams combined with β-lactamase inhibitors as a potential approach for treating infection of V. parahaemolyticus. The residues around the active site show certain variations, which can be useful for specific inhibitor design. In the directed evolution experiment, CARB-20 exhibited plasticity in developing significant resistance to inhibitors by accumulated residue substitutions. Therefore, careful monitoring of enzyme mutations is necessary for successfully applying β-lactam/β-lactamase inhibitor combination therapy. Taken together, our results open up an avenue of inhibitor design targeting vibrio β-lactamases, facilitating the application of β-lactams in treating vibrio infections.

Pseudomonas aeruginosa is a bacterial pathogen that causes severe chronic infections in immunocompromised individuals. This bacterium is highly adaptable to its environments, which frequently select for traits that promote bacterial persistence. A clinically significant temporal adaptation is the formation of surface- or cell-adhered bacterial biofilms that are associated with increased resistance to immune and antibiotic clearance. Extensive research has shown that bacterial flagellar motility promotes formation of such biofilms, whereupon the bacteria subsequently become nonmotile. However, recent evidence shows that antibiotic-tolerant nonattached bacterial aggregates, distinct from surface-adhered biofilms, can form, and these have been reported in the context of lung infections, otitis media, nonhealing wounds, and soft tissue fillers. It is unclear whether the same bacterial traits are required for aggregate formation as for biofilm formation. In this report, using isogenic mutants, we demonstrate that P. aeruginosa aggregates in liquid cultures are spontaneously formed independent of bacterial flagellar motility and independent of an exogenous scaffold. This contrasts with the role of the flagellum to initiate surface-adhered biofilms. Similarly to surface-attached biofilms, these aggregates exhibit increased antibiotic tolerance compared to planktonic cultures. These findings provide key insights into the requirements for aggregate formation that contrast with those for biofilm formation and that may have relevance for the persistence and dissemination of nonmotile bacteria found within chronic clinical infections. IMPORTANCE In this work, we have investigated the role of bacterial motility with regard to antibiotic-tolerant bacterial aggregate formation. Previous work has convincingly demonstrated that P. aeruginosa flagellar motility promotes the formation of surface-adhered biofilms in many systems. In contrast, aggregate formation by P. aeruginosa was observed for nonmotile but not for motile cells in the presence of an exogenous scaffold. Here, we demonstrate that both wild-type P. aeruginosa and mutants that genetically lack motility spontaneously form antibiotic-tolerant aggregates in the absence of an exogenously added scaffold. Additionally, we also demonstrate that wild-type (WT) and nonmotile P. aeruginosa bacteria can coaggregate, shedding light on potential physiological interactions and heterogeneity of aggregates.

A method to selectively enumerate Pseudomonas fluorescens from fresh chicken carcasses in less than 24 h using capacitance microbiology was developed. Capacitance assays were conducted on whole-carcass rinses at 25°C using brain heart infusion broth (BHI) containing 25 μg of Irgasan per ml to obtain a detection time. The capacitance samples were spread plated on plate count agar for isolation and identification. From plates with the highest dilution, from each carcass, 4 colonies were randomly selected and identified. Seven species of bacteria including Pseudomonas fluorescens were responsible for capacitance detection times. Various antibiotics and chemicals were added to basal media or brain heart infusion broth with Irgasan and were evaluated to select for the growth of P. fluorescens . BHI broth containing 4 μg of nitrofurantoin, 120 μg of carbenicillin, and 25 μg of Irgasan, all per ml, was found to be optimal and was termed Pseudomonas fluorescens selective additive (PSA) (patent pending). In a second study, 12 carcasses were collected in each of three replicate trials. For each trial, 2 carcasses were sampled immediately and 2 were sampled after storage at 3°C on days 3, 6, 9, 12, and 15. The BHI-PSA broth was found to be excellent for enumeration of P. fluorescens from broiler chicken carcass rinses in assays using capacitance microbiology at 25°C. The time required to enumerate P. fluorescens for all samples (day 0 to 15) was

The green iguana appears to be a reservoir for bacteria causing gastrointestinal infections in humans. The presence of diarrheogenicE. coli(DEC) pathotypes, however, has not been studied in this reptile. The aim of the current work was to investigate the prevalence of DEC in the intestines of 240 captive green iguanas, their phylogenetic groups, and the antibiotic susceptibility profile.E. colistrains were isolated from 41.7% of the intestinal content of green iguanas. DEC strains was identified in 25.9% of the screened population and were detected in the majority (62%, p=0.009] of those reptiles carryingE. colistrains. Among DEC strains, STEC strains carrying thestx1gene were the most prevalent pathotype isolated (38.7%), followed by EAEC and ETEC (27.4% each). Genetic markers of DEC strains belonging to the EHEC pathotype were not detected. More than a half of DEC strains were classified into the Clade I-II phylogroup (64.5%), followed by the phylogroup A (14.5%). The antibiotic susceptibility method demonstrated that a high proportion of DEC strains were resistance, or non-susceptible, to carbenicillin, amikacin, and ampicillin (85, 74, and 66%, respectively). We conclude that the green iguana kept in captivity is a reservoir of DEC strains bearing resistance to first-line antibiotics, including penicillins. Given the increase presence of the green iguana in Latin American households, these reptiles represent a potential source of transmission to susceptible humans and therefore a potential source of gastrointestinal disease.ImportanceLatin-American countries present a high burden of diarrheal disease. In this part of the world, besides common pathogens, diarrheal diseases are also caused by pathogenic types ofE. colireferred as “diarrheagenicE. coli” (DEC). While inhabitants of Latin American countries suffer of self-limiting diarrhea when infected with DEC, a main target of these strains are tourists from developed countries who are not exposed to DEC strains. Efforts are in place to decrease the burden of DEC-associated diarrheal disease. As such, this study investigated a potential reservoir of DEC strains that had been underestimated, the green iguana. These reptiles are very common in Latin American households and are found virtually everywhere in Mexico, Central and South America. We found that 25% of green iguanas carried DEC strains in their intestines. We also demonstrated a high prevalence of antibiotic resistance in these strains, posing a potential risk to humans.

Functional association between genomic loci and specific biological traits remains lacking in many fungi, including the African tree pathogen Ceratocystis albifundus. This is mainly because of the absence of suitable transformation systems for allowing genetic manipulation of this and other fungi. Here, we present an optimized protocol for Agrobacterium tumefaciens-mediated transformation of C. albifundus. Strain AGL-1 of A. tumefaciens and four binary T-DNA vectors (conferring hygromycin B or geneticin resistance and/or expressing the green fluorescent protein [GFP]) were used for transforming germinated conidia of three isolates of C. albifundus. Stable expression of these T-DNA-encoded traits was confirmed through sequential sub-culturing of fungal transformants on selective and non-selective media and by using PCR and sequence analysis. Single-copy integration of the respective T-DNAs into the genomes of these fungi was confirmed using Southern hybridization analysis. The range of experimental parameters determined and optimised included: (i) concentrations of hygromycin B and geneticin required for inhibiting growth of the wild type fungus and (ii) the dependence of transformation on acetosyringone for inducing the bacterium's virulence genes, as well as (iii) the duration of fungus-bacterium co-cultivation periods and (iv) the concentrations of fungal conidia and bacterial cells used for the latter. The system developed in this study is stable with a high-efficiency, yielding up to 400 transformants per 106 conidia. This is the first report of a transformation protocol for C. albifundus and its availability will be invaluable for functional studies in this important fungus.

The emerging and increasing prevalence of bacterial antibiotic resistance is a significant public health challenge. To begin to tackle this problem, it will be critical to not only understand the origins of this resistance but also document environmental reservoirs of antibiotic resistance. In this study we investigated the possibility that both colony and field caught mosquitoes could harbor antibiotic resistant bacteria. Specifically, we characterized the antibiotic resistant bacterial populations from colony-reared Aedes aegypti larvae and adults and two field caught mosquito species Coquillettidia perturbans and Ochlerotatus canadensis. The cultured bacterial populations were dominated by isolates belonging to the class Gammaproteobacteria. Among the antibiotic resistant populations, we found bacteria resistant to carbenicillin, kanamycin, and tetracycline, including bacteria resistant to a cocktail of all three antibiotics in combination. The antibiotic resistant bacteria were numerically rare, at most 5% of total cell counts. Isolates were characterized by 16S rRNA gene sequencing, and clustering into Operational Taxonomic Units (OTUs; 99% sequence identity). 27 antibiotic resistant OTUs were identified, although members of an OTU did not always share the same resistance profile. This suggests the clustering was either not sensitive enough to distinguish different bacteria taxa or different antibiotic resistant sub-populations exist within an OTU. Finally, the antibiotic selection opened up a niche to culture mosquito-associated fungi, and 10 fungal OTUs (28S rRNA gene sequencing) were identified. Two fungal OTUs both classified to the class Microbotryomycetes were commonly identified in the field-caught mosquitoes. Thus, in this study we demonstrate that antibiotic resistant bacteria and certain fungi are common and conserved mosquito microbiome members. These observations highlight the potential of invertebrates to serve as vehicles for the spread of antibiotic resistance throughout the environment.

The retrieval of a polystyrene macro-plastic piece stranded on the shores in King George Island (South Shetlands, Antarctica) gave the opportunity to explore the associated bacterial flora. A total of 27 bacterial isolates were identified by molecular 16s rRNA gene sequencing and 7 strains were selected and screened for their ability to produce biofilm and antibiotic susceptibility profiles. All the bacterial isolates were able to produce biofilm. The Kirby-Bauer disk diffusion susceptibility test to 34 antibiotics showed multiple antibiotic resistances against the molecules cefuroxime and cefazolin (belonging to cephalosporins), cinoxacin (belonging to quinolones) and ampicillin, amoxicillin + clavulanic acid, carbenicillin and mezlocillin (belonging to beta-lactams). The obtained results suggest that plastics can serve as vectors for the spread of multiple resistances to antibiotics across Antarctic marine environments and underline the relevance of future studies on this topic.

Halogenated 4-hydroxybenzylidene indolinones have been shown to re-sensitize methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecalis (VRE) to methicillin and vancomycin respectively. The mechanism of antibiotic re-sensitization was however not previously studied. Here, we probe the scope of antibiotic re-sensitization and present the global proteomics analysis of S. aureus treated with GW5074, a 4-hydroxybenzylidene indolinone compound. With a minimum inhibitory concentration (MIC) of 8 μg/mL against S. aureus, GW5074 synergized with beta-lactam antibiotics like ampicillin, carbenicillin and cloxacillin, the DNA synthesis inhibitor, ciprofloxacin, the protein synthesis inhibitor, gentamicin and the folate acid synthesis inhibitor, trimethoprim. Global proteomics analysis revealed that GW5074 treatment resulted in significant downregulation of enzymes involved in the purine biosynthesis. S. aureus proteins involved in amino acid metabolism and peptide transport were also observed to be downregulated. Interestingly, anti-virulence targets such as AgrC (a quorum sensing-related histidine kinase), AgrA (a quorum sensing-related response regulator) as well as downstream targets, such as hemolysins, lipases and proteases in S. aureus were also downregulated by GW5074. We observed that the peptidoglycan hydrolase, SceD was significantly upregulated. The activity of GW5074 on S. aureus suggests that the compound primes bacteria for the antibacterial action of ineffective antibiotics. Significance Antibiotic resistance continues to present significant challenges to the treatment of bacterial infections. Given that antibiotic resistance is a natural phenomenon and that it has become increasingly difficult to discover novel antibiotics, efforts to improve the activity of existing agents are worth pursuing. A few small molecules that re-sensitize resistant bacteria to traditional antibiotics have been described but the molecular details that underpin how these compounds work to re-sensitize bacteria remain largely unknown. In this report, global label-free quantitative proteomics was used to identify changes in the proteome that occurs when GW5074, a compound that re-sensitize MRSA to methicillin, is administered to S. aureus. The identification of pathways that are impacted by GW5074 could help identify novel targets for antibiotic re-sensitization.

Background: Urinary tract infection (UTI) is a mainly common infection in kidney transplant recipients. This study decided to investigate UTI, bacterial agents, and antibiotic resistance pattern in kidney transplant recipients from Iran. Materials and Methods: Search process was conducted for UTI, bacterial agents, and antibiotic resistance pattern in kidney transplant recipients from Iran via electronic databases (Scopus, PubMed, Web of Science, etc.,) with Mesh terms in either Persian and English languages without limited time to May 31, 2020. Data were analyzed by comprehensive meta-analysis software. Results: The combined prevalence of UTI in renal transplant recipients was reported by 31.1%. The combined prevalence of Gram-negative bacteria was 69%. The most common pathogens among Gram negatives were E. coli followed by Klebsiella pneumoniae with frequency 43.4% and 13%, respectively. Subgroup analysis for Gram-positive bacteria showed the combined prevalence of 31%. The most common microorganism among Gram positives belonged to coagulase-negative Staphylococci and Enterococci with a prevalence of 10.2% and 9%, respectively. Subgroup meta-analysis of antibiotic resistance for Gram-negative showed the most resistance to cephalexin followed by carbenicillin with a prevalence of 89.1% and 87.3%, respectively. Conclusion: Our review showed a noticeable rate of UTI (31.1%) among renal transplant recipients in Iran and a high prevalence of Gram-negative (69%) and Gram-positive (13%) microorganisms. A high resistance rate was seen against almost all antibiotics used for the treatment of UTI. Therefore, empirical prescription of antibiotics should be avoided, and it should be based on data obtained from antibiogram tests.

Introduction Multidrug-resistant (MDR) bacteria is a public health threat, which requires the development of new therapeutic options. The combined use of plant extracts with commercial antibiotics is an alternative against infections by MDR bacteria. Echeveria subrigida is a Crassulaceae plant with very good activity against some bacteria. This study aims to evaluate the synergistic effect of the methanol extract of Echeveria subrigida leaves (ME-ES) with commercial antibiotics against MDR isolates of Escherichia coli and Staphylococcus aureus . Methods Six antibiotics, E. coli (4 MDR isolates and ATCC 25922) and S. aureus (2 MDR isolates and ATCC 29213) were used. ATCC strains were susceptible to the corresponding antibiotics and used as controls. The minimal inhibitory (MIC) and bactericidal (MBC) concentrations were evaluated by the microdilution broth assay, and the synergistic effect by the checkerboard and the time-kill curve methods. Results The MDR of the bacterial isolates was corroborated, they were resistant to at least two families of antibiotics. The activity of ME-ES was good against one MDR E. coli isolate (MIC = 250 μg/mL) and the S. aureus strains (MIC = 250–1000 μg/mL), which was better than those registered for some commercial antibiotics. Synergism against S. aureus was found for the combinations ME-ES with carbenicillin and ME-ES with methicillin (FICI = 0.28 to 0.5). Conclusions ME-ES was active against S. aureus and increased its activity when combined with betalactamic antibiotics. ME-ES can contribute to providing a best treatment for infectious diseases caused by MDR S. aureus .

The study was undertaken to determine the pathogenic potential of Aeromonas spp.; Aeromonas hydrophila (18 isolates), Aeromonas veronii biovar sobria (15 isolates), and Aeromonas veronii biovar veronii (28 isolates) isolated from milk fish packed under modified atmosphere and vacuum and stored at 0–2 °C. The isolates were identified by biochemical methods and checked for the presence of 11 virulence genes; ahh1, aerA, act, ast, alt, asaI ahpA, ahpB, gcat, lip, and DNase. All the virulence genes were present in 39 % of the A. hydrophila isolates. Virulence-associated phenotypes were present in most of the isolates. The antibiotic resistance patterns of the Aeromonas spp. from fish revealed that all the isolates were resistance to ampicillin and penicillin. In addition, resistance of the isolates to cephalothin (86.7 %), carbenicillin (62.2 %), and erythromycin (40 %) was also observed. To our knowledge, this is the first report of isolation and characterisation of pathogenic Aeromonas spp. from modified atmosphere packed fish in India. The results of this study add to our knowledge of the ecology of this organism in packed fish, and may in the future help prevent infections in humans.

The wound healing process is frequently associated with a number of major clinical challenges, due to the failure of commonly used antibiotics as a remedy for wounds. There have always been fascinating questions about the novel applications of bioactive glasses (BGs) and it is expected that in the next few years these types of materials may play an important role in many aspects of soft tissue regeneration. This research focuses on the feasibility of using silver- and fluoride-containing BGs against multidrug-resistant bacterial strains isolated from patients with burns. According to the results obtained, fluoride did not exhibit antibacterial activity against the tested bacteria, while both 1% and 2% silver-containing BGs inhibited the bacterial growth. It is an important finding that 1% silver-containing BGs showed a potential antibacterial activity without any toxicity against fibroblasts, suggesting that this class of BGs could play a key role in the prevention of infection, reduction of pain, and removal of excessive exudates.

This research was conducted to develop genetic transformation of the recalcitrant chrysanthemum cv. Shinma by application of appropriate antibiotics and selective agents. Clavamox had the least inhibitory effect on shoot regeneration compared to timentin, carbenicillin, and cefotaxime. Clavamox, at a concentration of 125 mg L−1, was found to be the most suitable for shoot regeneration and production of quality shoots, suppressing the growth of Agrobacterium in explants infected with strains GV3101 or C58C1 for 3 and 4 weeks, respectively. The concentration of phosphinothricin (PPT) was found to be 1.0 mg L−1 for screening of putative transgenic shoots. Moreover, transgenic chrysanthemums were obtained by culturing explants co-cultivated with A. tumefaciens strain GV3101 harboring an anthocyanin regulatory gene RsMYB1 isolated from radish (Raphanus sativus), which was placed under the control of cauliflower mosaic virus promoter (CaMV) 35S and petal-specific promoter InMYB1 isolated from the mornin...

Background/aims Cronobacter sakazakii, an emergent pathogen is considered as a major concern to infants and neonates fed on reconstituted powdered infant milk formula. In conjunction with many other factors, biofilm forming capacity adds to its pathogenic potential. In view of the facts that infants are at highest risk to C. sakazakii infections, and emerging antibiotic resistance among pathogens, it is imperative to evaluate probiotic cultures for their efficacy against C. sakazakii. Therefore, pure probiotic strains were isolated from commercial probiotic products and tested for their antimicrobial and anti-biofilm activities against C. sakazakii. Methods A total of 6 probiotic strains were tested for their antibiotic susceptibility followed by antimicrobial activity using cell-free supernatant (CFS) against C. sakazakii. The inhibitory activity of CFS against biofilm formation by C. sakazakii was determined using standard crystal violet assay and microscopic observations. Results All the probiotic strains were sensitive to ampicillin, tetracycline, vancomycin and carbenicillin whereas most of the strains were resistant to erythromycin and novobiocin. Four of the 6 probiotic derived CFS possessed antimicrobial activity against C. sakazakii at a level of 40 μL. A higher biofilm inhibitory activity (>80%) was observed at initial stages of biofilm formation with weaker activity during longer incubation upto 48 hours (50%-60%). Conclusions The study indicated the efficacy of isolated commercial probiotics strains as potential inhibitor of biofilm formation by C. sakazakii and could be further explored for novel bioactive molecules to limit the emerging infections of C. sakazakii.

Rapid and sensitive detection of mycobacteria is essential for the successful treatment and control of mycobacterial infections. It is considered good laboratory practice to combine a liquid medium with at least one solid medium for culturing mycobacteria from clinical specimens. Automated systems with liquid media provide faster results and are more sensitive, while solid media help to detect mixed infections and can detect mycobacteria that do not grow in the broth medium. In order to evaluate the BacT/ALERT 3D system (bioMerieux, Durham, NC, USA) and to find the optimal combination of media, we compared the results achieved with BacT/ALERT 3D, Lowenstein-Jensen (L-J) medium (Bio-Rad, Marne-la Coquette, France), and the Middlebrook 7H10/7H11 biplate (Becton Dickinson, Sparks, MD, USA) on consecutive clinical specimens collected within a period of 26 months. A total of 6,250 clinical samples (4,051 respiratory specimens, 1,209 normally sterile body fluids, 292 biopsy specimens, 247 blood specimens, 344 urine specimens and 304 other specimens) from 3,247 patients were tested for the presence of mycobacteria. Specimens with a different panel of media (blood and urine) and specimens contaminated with bacteria other than mycobacteria were excluded from the evaluation. Tissue specimens were crushed and homogenized. Homogenized tissue, respiratory specimens and other specimens from contaminated sites were decontaminated with sodium dodecyl sulfate-NaOH as described by Tacquet and Tisson [1]. Liquids from sterile sites were concentrated by centrifugation (3000 g, 15 min). Aliquots of 0.3–0.5 ml were inoculated onto a L-J slant, a Middlebrook 7H10/7H11 selective agar biplate with carbenicillin, polymyxin B, trimethoprim lactate, and amphotericin B, and into a BacT/ALERT MP bottle. For specimens from

Bacteria commonly live in dense and genetically diverse communities associated with surfaces. In these communities, competition for resources and space is intense, and yet we understand little of how this affects the spread of antibiotic- resistant strains. Here, we study interactions between antibiotic-resistant and susceptible strains using in vitro competition experiments in the opportunistic pathogen Pseudomonas aeruginosa and in silico simulations. Selection for intracellular resistance to streptomycin is very strong in colonies, such that resistance is favoured at very low antibiotic doses. In contrast, selection for extracellular resistance to carbenicillin is weak in colonies, and high doses of antibiotic are required to select for resistance. Manipulating the density and spatial structure of colonies reveals that this difference is partly explained by the fact that the local degradation of carbenicillin by β-lactamase-secreting cells protects neighbouring sensitive cells from carbenicillin. In addition, we discover a second unexpected effect: the inducible elongation of cells in response to carbenicillin allows sensitive cells to better compete for the rapidly growing colony edge. These combined effects mean that antibiotic treatment can select against antibiotic-resistant strains, raising the possibility of treatment regimes that suppress sensitive strains while limiting the rise of antibiotic resistance. We argue that the detailed study of bacterial interactions will be fundamental to understanding and overcoming antibiotic resistance.

It is widely acknowledged that faster-growing bacteria are killed faster by β-lactam antibiotics. This notion serves as the foundation for the concept of bacterial persistence: dormant bacterial cells that do not grow are phenotypically tolerant against β-lactam treatment. Such correlation has often been invoked in the mathematical modeling of bacterial responses to antibiotics. Due to the lack of thorough quantification, however, it is unclear whether and to what extent the bacterial growth rate can predict the lysis rate upon β-lactam treatment under diverse conditions. Enabled by experimental automation, here we measured >1,000 growth/killing curves for eight combinations of antibiotics and bacterial species and strains, including clinical isolates of bacterial pathogens. We found that the lysis rate of a bacterial population linearly depends on the instantaneous growth rate of the population, regardless of how the latter is modulated. We further demonstrate that this predictive power at the population level can be explained by accounting for bacterial responses to the antibiotic treatment by single cells. This linear dependence of the lysis rate on the growth rate represents a dynamic signature associated with each bacterium–antibiotic pair and serves as the quantitative foundation for designing combination antibiotic therapy and predicting the population-structure change in a population with mixed phenotypes.

Vibrio parahaemolyticus is a Gram-negative halophilic marine microbe that causes gastroenteritis, wound infections, and septicemia in humans. Since the emergence of the pandemic clone O3:K6, V. parahaemolyticus has become a globally well-known pathogen. In this study, 375 seawater samples collected from the Eastern coast of Saudi Arabia were tested for the presence of V. parahaemolyticus. Three hundred and forty samples were determined positive for V. parahaemolyticus using traditional microbiological techniques. The genes toxR and tlh were detected via polymerase chain reaction (PCR) in 41 isolates from 23 samples (6%). Thermostable direct hemolysin (tdh) and thermostable direct hemolysin-related hemolysin (tdh) are the most common virulence genes associated with V. parahaemolyticus. As such, four isolates were tdh+ (1%) and another four were trh+ (1%). No evidence of pandemic clones was detected using group-specific PCR (GS-PCR). Samples were tested for antibiotic susceptibility against 28 agents. The vast majority of samples exhibited high resistance to carbenicillin (98%), ampicillin (88%), and cephalothin (76%). The multiple antibiotics resistance index was >0.2 for 35% of the isolates. The results of this study confirm the presence of V. parahaemolyticus in the Eastern coast of Saudi Arabia. This is the first report of tdh+ and trh+ isolates from this area.

Biofilm formation is one of the many mechanisms bacteria utilize to survive antibiotic treatment. It has been demonstrated that when Mycobacterium tuberculosis exists in a biofilm in vitro it expresses phenotypic resistance to antimicrobial drugs. Since the in vivo survival of M. tuberculosis following drug treatment is potentially linked to a biofilm-like expression of drug tolerance, it is hypothesized that biofilm dispersion should increase antibiotic susceptibility and reduce the duration of the current antibiotic treatment regimen. Previously, we have identified a 2-aminoimidazole (2-AI) compound capable of dispersing and inhibiting M. tuberculosis and M. smegmatis biofilms in vitro. Additionally, this compound potentiated the activity of carbenicillin against M. tuberculosis and, to a lesser degree, M. smegmatis. Here, we describe a SAR study on this compound evaluating each derivative for biofilm dispersion and β-lactam potentiation capabilities against M. smegmatis. This study identified a compound that improved upon the biofilm dispersion capabilities of the lead compound. Interestingly, a different compound was identified with an increased ability to potentiate a subset of β-lactam antibiotics. These compounds indicate that biofilm dispersion and potentiation capabilities may not be associated.

Here, the influence of metabolizable sugars on the susceptibility of Escherichia coli to β-lactam antibiotics was investigated. Notably, monitoring growth and survival of mono- and combination-treated planktonic cultures showed a 1000- to 10 000-fold higher antibacterial efficacy of carbenicillin and cefuroxime in the presence of certain sugars, whereas other metabolites had no effect on β-lactam sensitivity. This effect was unrelated to changes in growth rate. Light microscopy and flow cytometry profiling revealed that bacterial filaments, formed due to β-lactam-mediated inhibition of cell division, rapidly appeared upon β-lactam mono-treatment and remained stable for up to 18 h. The presence of metabolizable sugars in the medium did not change the rate of filamentation, but led to lysis of the filaments within a few hours. No lysis occurred in E. coli mutants unable to metabolize the sugars, thus establishing sugar metabolism as an important factor influencing the bactericidal outcome of β-lactam treatment. Interestingly, the effect of sugar on β-lactam susceptibility was suppressed in a strain unable to synthesize the nutrient stress alarmone (p)ppGpp. Here, to the best of our knowledge, we demonstrate for the first time a specific and significant increase in β-lactam sensitivity due to sugar metabolism in planktonic, exponentially growing bacteria, unrelated to general nutrient availability or growth rate. Understanding the mechanisms underlying the nutritional influences on antibiotic sensitivity is likely to reveal new proteins or pathways that can be targeted by novel compounds, adding to the list of pharmacodynamic adjuvants that increase the efficiency and lifespan of conventional antibiotics.

Chromobacterium violaceum is an opportunistic pathogen that causes infections that are difficult to treat. The goal of this research was to evaluate the effect of selected tannins (tannic acid (TA) and gallic acid (GA)) on bacterial growth, motility, antibiotic (carbenicillin, tetracycline) susceptibility, and biofilm formation. Both tannins, particularly TA, impaired bacterial growth levels and swimming motilities at sub-minimum inhibitory concentrations (sub-MICs). In combination with tannins, antibiotics showed increased MICs, suggesting that tannins interfered with antibacterial activity. Sub-MICs of tetracycline or TA alone enhanced biofilm formation of C. violaceum; however, in combination, these compounds inhibited biofilm formation. In contrast, carbenicillin at sub-MICs was effective in inhibiting C. violaceum biofilm formation; however, in combination with lower concentrations of TA or GA, biofilms were enhanced. These results provide insights into the effects of tannins on C. violaceum growth and their varying interaction with antibiotics used to target C. violaceum infections.

An Agrobacterium-mediated transformation method was applied to introduce the luciferase reporter gene under the control of the CaMV35S promoter in the pGreen0049 binary vector into strawberry cv. Camarosa. The in vitro regeneration system of strawberry leaves to be used in the transformation was optimized using different TDZ concentrations in MS medium. TDZ at 16 µM showed the highest percentage (100%) of shoot formation and the highest mean number of shoots (24) produced per explant. Studies on the effects of different antibiotics, namely timentin, cefotaxime, carbenicillin and ampicillin, on shoot regeneration of strawberry leaf explants showed the best shoot regeneration in the presence of 300 mg/L timentin and 150 mg/L cefotaxime. Assessment of the different factors affecting Agrobacterium mediated-transformation of strawberry with the luciferase gene showed the highest efficiency of putative transformant production (86%) in the treatment with no preculture, bacterial OD600 of 0.6 and the addition of 150 mg/L cefotaxime in the pre-selection and selection media. The presence of the luciferase gene in the plant genome was verified by the luciferase reporter gene assay, nested PCR amplification and dot blot of genomic DNA isolated from the young leaves of each putatively transformed plantlet.

ICE1 genes play a very important role in plants in cold conditions. To improve the cold resistance of tomato, the ICE1 gene of Arabidopsis thaliana was used to construct the plant expression vector p3301-ICE1, and was overexpressed in tomato through Agrobacterium-mediated transformation. Five strains of resistant plants were obtained. PCR and half-quantitative results showed that the ICE1 gene was transferred to tomato; three strains tested positive. After low-temperature stress treatment, praline content and peroxide and catalase activities in the transgenic tomato plants were higher compared with non-transgenic controls, while malondialdehyde content was clearly lower.

Steroids, including testosterone, estrone, 17β-estradiol, estriol and 17β-ethinyl estradiol, are harmful not only to the population dynamics of aquatic life forms but also to public health. In this study, a marine testosterone-degrading bacterium (strain N3) was isolated from Nanao Island in the South China Sea. In addition, the strain could also use 17β-estradiol (E2), 17β-ethinyl estradiol (EE2), estriol (E3) or cholesterol as a sole carbon source. According to the 16S rRNA gene sequence analysis, strain N3 was identified as Vibrio sp. Further characterization showed that the strain is aerobic, gram-negative, and mobile and exhibits resistance to ampicillin, carbenicillin, penicillin and spectinomycin. For enhancing its capacity of testosterone degradation, the Plackett-Burman factorial design and the central composite design were used to optimize the culture condition. Under optimal conditions, 92% of testosterone was degraded by Vibrio sp. N3 in 48h.