Major Gram-negative Pathogens Research Articles

Antimicrobial resistance survey and whole-genome analysis of nosocomial P. Aeruginosa isolated from eastern Province of China in 2016–2021

BackgroundPseudomonas aeruginosa is a major Gram-negative pathogen that can exacerbate lung infections in the patients with cystic fibrosis, which can ultimately lead to death.MethodsFrom 2016 to 2021, 103 strains of P. aeruginosa were isolated from hospitals and 20 antibiotics were used for antimicrobial susceptibility determination. Using next-generation genome sequencing technology, these strains were sequenced and analyzed in terms of serotypes, ST types, and resistance genes for epidemiological investigation.ResultsThe age distribution of patients ranged from 10 days to 94 years with a median age of 69 years old. The strains were mainly isolated from sputum (72 strains, 69.9%) and blood (14 strains, 13.6%). The size of these genomes ranged from 6.2 Mb to 7.4 Mb, with a mean value of 6.5 Mb. In addition to eight antibiotics that show inherent resistance to P. aeruginosa, the sensitivity rates for colistin, amikacin, gentamicin, ceftazidime, piperacillin, piperacillin-tazobactam, ciprofloxacin, meropenem, aztreonam, imipenem, cefepime and levofloxacin were 100%, 95.15%, 86.41%, 72.82%, 71.84%, 69.90%, 55.34%, 52.43%, 50.49%, 50.49%, 49.51% and 47.57% respectively, and the carriage rate of MDR strains was 30.69% (31/101). Whole-genome analysis showed that a total of 50 ST types were identified, with ST244 (5/103) and ST1076 (4/103) having a more pronounced distribution advantage. Serotype predictions showed that O6 accounted for 29.13% (30/103), O11 for 23.30% (24/103), O2 for 18.45% (19/103), and O1 for 11.65% (12/103) of the highest proportions. Notably, we found a significantly higher proportion of ExoU in P. aeruginosa strains of serotype O11 than in other cytotoxic exoenzyme positive strains. In addition to this, a total of 47 crpP genes that mediate resistance to fluoroquinolones antibiotics were found distributed on 43 P. aeruginosa strains, and 10 new variants of CrpP were identified, named 1.33, 1.34, 1.35, 1.36, 1.37, 1.38, 1.39, 1.40, 1.41 and 7.1.ConclusionsWe investigated the antibiotic susceptibility of clinical isolates of P. aeruginosa and genomically enriched the diversity of P. aeruginosa for its prophylactic and therapeutic value.

In vivo functional immunoprotection correlates for vaccines against invasive bacteria

Vaccination has significantly reduced the incidence of invasive infections caused by several bacterial pathogens, including Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria meningitidis. However, no vaccines are available for many other invasive pathogens. A major hurdle in vaccine development is the lack of functional markers to quantify vaccine immunity in eliminating pathogens during the process of infection. Based on our recent discovery of the liver as the major organ of vaccine-induced clearance of blood-borne virulent bacteria, we here describe a new vaccine evaluation system that quantitatively characterizes the key features of effective vaccines in shuffling virulent bacteria from the blood circulation to the liver resident macrophage Kupffer cells (KCs) and sinusoidal endothelial cells (LSECs) in mouse septic infection model. This system consists of three related correlates or assays: pathogen clearance from the bloodstream, pathogen trapping in the liver, and pathogen capture by KCs/LSECs. These readouts were consistently associated with the serotype-specific immunoprotection levels of the 13-valent pneumococcal polysaccharide conjugate vaccine (PCV13) against lethal infection of S. pneumoniae, a major invasive Gram-positive pathogen of community-acquired infections in humans. Furthermore, the reliability and sensitivity of these correlates in reflecting vaccine efficacy were verified with whole cell vaccines of Klebsiella pneumoniae and Escherichia coli, two major Gram-negative pathogens in hospital-acquired invasive infections. This system may be used as effective readouts to evaluate the immunoprotective potential of vaccine candidates in the preclinical phase by filling the current technical gap in vaccine evaluation between the conventional in vitro approaches (e.g. antibody production and pathogen neutralization/opsonophagocytosis) and survival of immunized animals.

Evaluation of antibacterial activity of pomegranate (Punica granatum L. and Citrus lemon against urinary tract infection causing bacteria E. Coli, Enterococcus fecalis, Enterobacter aerogenes collected from various diagnostic laboratories of Khwazakhela,

E. Coli is one of the major gram-negative pathogens well known causative agent for almost all kind of UTIs, however Enterobacteriaceae such as Enterobacter aerogenes and Enterococci fecalis are responsible for UT infections. The present study was conducted to evaluate the in vitro antibacterial activity of ethanol and aqueous extract of pomegranate and lemon peels against (23 isolated stains) E. Coli, (16 isolated strains) Enterococcus faecalis and (9 isolated strain) of Enterobactor aerogenes recovered from the urine sample of patientsin Swat KPK-Pakistan. The Agar dilution and well method was applied for the antibacterial activity of pomegranate and lemon peelextracts. All of the three subject isolates were exposed to different concentrations of pomegranate and lemon peel. MIC of aqua extract of pomegranateagainst the subject isolates of E. Coli was 200 µl/mLand ethanol extract was 50 µl/mL. So the ethanol extract of pomegranate was found to be more effective as compared to the aqueous extract. On the other hand MIC of aqua extract lemon peel against the subject isolates of E. coli was100 µl/mL and ethanol extract was 25 µl/mL. So the ethanol extract of pomegranate was found to be more effective as compared to the aqueous extract. MIC of ethanol extract of pomegranate against the isolates of Enterococcus fecalis was found smaller (50 µl/mL) than that of water extract (100 µl/mL). So the ethanol extract of pomegranate was found to be more effective as compared to the aqueous extract. On the other hand. MIC of aqua lemon extract against the subject isolates of Enterococcus fecalis was 100 µl/mL and ethanol extract was 25 µl/mL. Mainly the ethanolic extract of lemon peel was more effective than the ethanolic extract of pomegranate against Enterococcus fecalis isolates. The MIC of the water and ethanolic extract of pomegranate against Enterobacter aerogenes was found 500 µl/mL and 200 µl/mL respectively. So the ethanol extract of pomegranate was found to be more effective as compared to the aqueous extract. On the other hand MIC of the ethanolic extract of lemon peel was 100 µl/mL, while the water extracts showed inhibition against the Enterobacter aerogenes isolates at the concentration of 250 µl/mL. So the ethanol extract of lemon was found to be more effective as compared to the aqueous extract.For the determination of antibacterial activity of both pomegranate and lemon extracts (in ethanol and water) method as described in Clinical and Laboratory Standard Guidelines (CLSI) was adopted. The subject isolate of E. Coli, Enterococcus fecali, and Enterobacter aerogenes were shown different sensitivities (sensitive >10mm zone inhibition or resistant <10mm zone inhibition t) for ciprofloxacin with a variable zone of inhibition.

Systemic Administration of Porphyromonas Gingivalis Lipopolysaccharide Induces Glial Activation and Depressive-Like Behavior in Rats.

Periodontitis is one of the most common chronic inflammatory disorders in adults. Although clinical studies have suggested a causal relationship between periodontitis and major depression (MD), the biological mechanisms by which periodontitis instigates MD are unknown. We investigated whether a systemic administration of lipopolysaccharide (LPS) from Porphyromonas gingivalis (Pg), a major Gram-negative pathogen of periodontitis, causes depressive-like behavior and glial activation in the hippocampus and the prefrontal cortex (PFC), which are MD-related brain regions. Eight-week-old male Sprague Dawley rats were randomly divided into a behavioral test group and an immunohistochemistry group. The rats in each group were further assigned to the sham injection (saline) and Porphyromonas gingivalis-lipopolysaccharide (Pg-LPS) injection protocols. The rats received an intraperitoneal injection of saline or Pg-LPS with gradually increasing doses (day 1: 0.5, day 2: 0.5, day 3: 0.75, day 4: 0.75, day 5: 1.0, day 6: 1.0, and day 7: 1.0 mg/kg of body weight) for seven consecutive days. After the systemic administration, the behavior test group underwent the forced swimming test (FST) and Y-maze test. For the immunohistochemistry group, we quantified the immunoreactivity for microglial Iba-1 (ionized calcium-binding adapter molecule 1) and astrocytic glial fibrillary acidic protein (GFAP) in the hippocampus (dentate gyrus [DG], cornu ammonis [CA1 and CA3]) and PFC (prelimbic [PrL] and the infralimbic [IL]) areas. The FST immobility time in the Pg-LPS group was significantly longer than that in the sham group. In the Y-maze test, a significant decline in spontaneous alternation behavior was observed in the Pg-LPS group compared to the sham group. The peripheral administration of Pg-LPS significantly increased the immunoreactivity for Iba-1 in the CA3 and PrL. Pg-LPS injection significantly increased the immunoreactivity for GFAP in the DG, CA1, and CA3. The major result of this study is that a repeated systemic administration of Pg-LPS caused depressive-like behavior and both microglial and astrocytic activation in rats. This finding may comprise biological evidence of a causal relationship between periodontitis and MD.

Antimicrobial Resistance in Pseudomonas aeruginosa before and during the COVID-19 Pandemic.

Pseudomonas aeruginosa (PA) is a major Gram-negative opportunistic pathogen causing several serious acute and chronic infections in the nosocomial and community settings. PA eradication has become increasingly difficult due to its remarkable ability to evade antibiotics. Therefore, epidemiological studies are needed to limit the infection and aim for the correct treatment. The present retrospective study focused on PA presence among samples collected at the San Giovanni di Dio and Ruggi D'Aragona University Hospital in Salerno, Italy; its resistance profile and relative variations over the eight years were analyzed. Bacterial identification and antibiotic susceptibility tests were performed by VITEK® 2. In the 2015-2019 and 2020-2022 timeframes, respectively, 1739 and 1307 isolates of PA were obtained from respiratory samples, wound swabs, urine cultures, cultural swabs, blood, liquor, catheter cultures, vaginal swabs, and others. During 2015-2019, PA strains exhibited low resistance against amikacin (17.2%), gentamicin (25.2%), and cefepime (28.3%); moderate resistance against ceftazidime (34.4%), imipenem (34.6%), and piperacillin/tazobactam (37.7%); and high resistance against ciprofloxacin (42.4%) and levofloxacin (50.6%). Conversely, during the 2020-2022 era, PA showed 11.7, 21.1, 26.9, 32.6, 33.1, 38.7, and 39.8% resistance to amikacin, tobramycin, cefepime, imipenem, ceftazidime, ciprofloxacin, and piperacillin/tazobactam, respectively. An overall resistance-decreasing trend was observed for imipenem and gentamicin during 2015-2019. Instead, a significant increase in resistance was recorded for cefepime, ceftazidime, and imipenem in the second set of years investigated. Monitoring sentinel germs represents a key factor in optimizing empirical therapy to minimize the spread of antimicrobial resistance.

Safety evaluations of a synthetic antimicrobial peptide administered intravenously in rats and dogs

The antimicrobial peptide SET-M33 is under study for the development of a new antibiotic against major Gram-negative pathogens. Here we report the toxicological evaluation of SET-M33 administered intravenously to rats and dogs. Dose range finding experiments determined the doses to use in toxicokinetic evaluation, clinical biochemistry analysis, necroscopy and in neurological and respiratory measurements. Clinical laboratory investigations in dogs and rats showed a dose-related increase in creatinine and urea levels, indicating that the kidneys are the target organ. This was also confirmed by necroscopy studies of animal tissues, where signs of degeneration and regeneration were found in kidney when SET-M33 was administered at the highest doses in the two animal species. Neurological toxicity measurements by the Irwin method and respiratory function evaluation in rats did not reveal any toxic effect even at the highest dose. Finally, repeated administration of SET-M33 by short infusion in dogs revealed a no-observed-adverse-effect-level of 0.5 mg/kg/day.

Anti-Virulence Activity of 3,3'-Diindolylmethane (DIM): A Bioactive Cruciferous Phytochemical with Accelerated Wound Healing Benefits.

Antimicrobial resistance is among the top global health problems with antibacterial resistance currently representing the major threat both in terms of occurrence and complexity. One reason current treatments of bacterial diseases are ineffective is the occurrence of protective and resistant biofilm structures. Phytochemicals are currently being reviewed for newer anti-virulence agents. In the present study, we aimed to investigate the anti-virulence activity of 3,3′-diindolylmethane (DIM), a bioactive cruciferous phytochemical. Using a series of in vitro assays on major Gram-negative pathogens, including transcriptomic analysis, and in vivo porcine wound studies as well as in silico experiments, we show that DIM has anti-biofilm activity. Following DIM treatment, our findings show that biofilm formation of two of the most prioritized bacterial pathogens Acinetobacter baumannii and Pseudomonas aeruginosa was inhibited respectively by 65% and 70%. Combining the antibiotic tobramycin with DIM enabled a high inhibition (94%) of P. aeruginosa biofilm. A DIM-based formulation, evaluated for its wound-healing efficacy on P. aeruginosa-infected wounds, showed a reduction in its bacterial bioburden, and wound size. RNA-seq was used to evaluate the molecular mechanism underlying the bacterial response to DIM. The gene expression profile encompassed shifts in virulence and biofilm-associated genes. A network regulation analysis showed the downregulation of 14 virulence-associated super-regulators. Quantitative real-time PCR verified and supported the transcriptomic results. Molecular docking and interaction profiling indicate that DIM can be accommodated in the autoinducer- or DNA-binding pockets of the virulence regulators making multiple non-covalent interactions with the key residues that are involved in ligand binding. DIM treatment prevented biofilm formation and destroyed existing biofilm without affecting microbial death rates. This study provides evidence for bacterial virulence attenuation by DIM.

Multidrug-resistant gram-negative bacterial infections the Tamale Teaching Hospital in Northern Ghana: a three-year retrospective analysis

Background: A variety of clinically important pathogens have developed multidrug resistance (MDR), which threatens global public health. This study aimed to determine the incidence, patterns, and trends of MDR of gram-negative bacterial isolates in clinical specimens in the Tamale Teaching Hospital, Ghana. Methods: This retrospective study analyzed gram-negative bacterial isolates and antimicrobial susceptibility test (AST) results of patients who visited the Tamale Teaching Hospital laboratory between 2017 and 2019. Results: A total of 2,779 gram-negative bacterial isolates and their phenotypic AST results were analyzed. From these, 1,297 gram-negative bacteria (46.7%) were isolated from urine samples, while the rest were isolated from sputum (20.9%), wound (14.3%), and swabs (11.7%) samples, etc. Escherichia coli (23.8%) was the most common gram-negative pathogen found predominantly in the urine samples (33.2%). All gram-negative bacteria isolated between 2017 and 2019 showed high MDR. Klebsiella pneumoniae gradually increased its MDR from 84.0% in 2017, 89.5% in 2018, to 95.1% in 2019. On the other hand, the MDR rates in Pseudomonas aeruginosa were approximately 65.8%, varying from 59.5% in 2017 to 78.7% in 2019. Among tested antimicrobials, amikacin was the most effective. Resistance to amikacin in Enterobacter spp., E. coli, and K. pneumoniae in vitro were 16.2%, 11.8%, and 17.7%, respectively. Conclusion: The study has shown that the high levels of MDR in gram-negative bacteria isolated may be associated with the infections recorded at the Tamale Teaching Hospital. The major gram-negative pathogens isolated have resistance to penicillins, cephalosporins, and fluoroquinolones. Aminoglycosides can offer high antibiotic activity to overcome gramnegative bacterial resistance. Further studies will be needed to decide policy direction on infection prevention and control, and antimicrobial stewardship programs.

Isolation of Metallo Beta Lactamases from Clinical Sample (MBL)

The rapid spread of acquired metallo-beta-lactamases (MBLs) among major Gram-negative pathogens is an emerging threat and a matter of particular concern worldwide. Carbapenems are among the few useful antibiotics against gram negative bacteria particularly those with extended spectrum beta-lactamase. Resistant to carbapenem is mediated by loss of outer membrane proteins and production of beta lactamase that is capable of hydrolyzing carbapenems.During this study period, 80 different clinical samples were collected from various hospitals of Raipur. All clinical samples were processed according to standard microbiological methods. Isolated GNRs were subjected to susceptibility testing against antibiotics by disc diffusion method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. Carbapenem-resistant isolates were subjected to the detection of MBL production by different methods. Out of 80 samples, 28 (35%) samples showed significant growth. MBL production was observed in 20 samples.

Prognostic role of time to positivity of blood culture in children with Pseudomonas aeruginosa bacteremia

BackgroundPseudomonas aeruginosa (P. aeruginosa) is a major Gram-negative pathogen, which has been reported to result in high mortality. We aim to investigate the prognostic value and optimum cut-off point of time-to-positivity (TTP) of blood culture in children with P. aeruginosa bacteremia.MethodsFrom August 2014 to November 2018, we enrolled the inpatients with P. aeruginosa bacteremia in a 1500-bed tertiary teaching hospital in Chongqing, China retrospectively. Receiver operating characteristic (ROC) analysis was used to determine the optimum cut-off point of TTP, and logistic regression were employed to explore the risk factors for in-hospital mortality and septic shock.ResultsTotally, 52 children with P. aeruginosa bacteremia were enrolled. The standard cut-off point of TTP was18 h. Early TTP (≤18 h) group patients had remarkably higher in-hospital mortality (42.9% vs 9.7%, P = 0.014), higher incidence of septic shock (52.4% vs12.9%, P = 0.06), higher Pitt bacteremia scores [3.00 (1.00–5.00) vs 1.00 (1.00–4.00), P = 0.046] and more intensive care unit admission (61.9% vs 22.6%, P = 0.008) when compared with late TTP (> 18 h) groups. Multivariate analysis indicated TTP ≤18 h, Pitt bacteremia scores ≥4 were the independent risk factors for in-hospital mortality (OR 5.88, 95%CI 1.21–21.96, P = 0.035; OR 4.95, 95%CI 1.26–27.50, P = 0.024; respectively). The independent risk factors for septic shock were as follows: TTP ≤18 h, Pitt bacteremia scores ≥4 and hypoalbuminemia (OR 6.30, 95%CI 1.18–33.77, P = 0.032; OR 8.15, 95%CI 1.15–42.43, P = 0.014; OR 6.46, 95% CI 1.19–33.19 P = 0.031; respectively).ConclusionsEarly TTP (≤18 hours) appeared to be associated with worse outcomes for P. aeruginosa bacteremia children.

Comprehensive Host Cell-Based Screening Assays for Identification of Anti-Virulence Drugs Targeting Pseudomonas aeruginosa and Salmonella Typhimurium.

The prevalence of bacterial pathogens being resistant to antibiotic treatment is increasing worldwide, leading to a severe global health challenge. Simultaneously, the development and approval of new antibiotics stagnated in the past decades, leading to an urgent need for novel approaches to avoid the spread of untreatable bacterial infections in the future. We developed a highly comprehensive screening platform based on quantification of pathogen driven host-cell death to detect new anti-virulence drugs targeting Pseudomonas aeruginosa (Pa) and Salmonella enterica serovar Typhimurium (ST), both known for their emerging antibiotic resistance. By screening over 10,000 small molecules we could identify several substances showing promising effects on Pa and ST pathogenicity in our in vitro infection model. Importantly, we could detect compounds potently inhibiting bacteria induced killing of host cells and one novel comipound with impact on the function of the type 3 secretion system (T3SS) of ST. Thus, we provide proof of concept data of rapid and feasible medium- to high-throughput drug screening assays targeting virulence mechanisms of two major Gram-negative pathogens.

Potential Inhibitors Against NDM-1 Type Metallo-β-Lactamases: An Overview.

A new member of the class metallo-β-lactamase (MBL), New Delhi metallo-beta-lactamase 1 (NDM-1) has emerged recently as a leading threat to the treatment of infections that have spread in all major Gram-negative pathogens. The enzyme inactivates antibiotics of the carbapenem family, which are a mainstay for the treatment of antibiotic-resistant bacterial infections. This review provides information about NDM-1 spatial structure, potential features of the active site, and its mechanism of action. It also enlists the inhibitors/compounds/drugs against NDM-1 in various development phases. Understanding their mode of inhibition and the structure-activity relationship would be beneficial for development, synthesis, and even increasing biological efficacy of inhibitors, making them more promising drug candidates.

Comparative analysis of efficiency of bacteriological and molecular-biological methods for the assessment of microbial contamination of hospital environment objects

Aim. To conduct a comparative analysis of bacteriological and molecular biological research methods to assess the microbial contamination of objects of hospital environment as factors of microbial transmission. Materials and methods. A comprehensive laboratory investigation of nosocomial environment of surgical, intensive care, hematology divisions of a large multi- disciplinary clinic during 2017 was carried out, while 215 samples were selected. Results. Microbial contamination of hospital environment facilities accounted for 54,0% when carrying out bacteriological tests and 80,0% — in conducting molecular biological studies. Between the frequency of bacteriological isolation of microorganisms and the determination of DNA of pathogens in different departments of the hospital, a strong direct correlation was revealed (r=0.92). The coincidence of the results of the two methods of research in most cases was observed at high values of the number of DNA copies (800-10000). The molecular biological method of diagnosis revealed the Methicillin-resistant Staphylococcus 1.9 times more often and in 19,4±7,2% lactamase (MBL) genes in major gram-negative pathogens. Conclusion. The use of molecular biological methods allows to detect microorganisms on the objects of hospital environment much more often than during bacteriological examination.

Multiple carbapenemase gene production by Acinetobacter baumannii isolates from burn patients in Iran

Objective: Acinetobacter baumannii is among the major Gram-negative nosocomial pathogens, and their antibiotic resistance has spread extensively; especially in burn settings. Methods: A total of 300 clinical isolates of A. baumannii were collected from burn patients hospitalized in burn settings. The isolates were examined for antimicrobial susceptibility testing by the disc diffusion method. The class D (bla(OXA-51)-like, bla(OXA-23)-like, bla(OXA-58)-like and bla(OXA-24)-like) and class B (encoded by bla(VIM) and bla(SIM)) carbapenemase genes were investigated by multiplex PCR. Results: A high level of carbapenem resistance was observed among isolates, but none of them were resistant to colistin. Among carbapenem-resistant A. baumannii, previous antibiotic consumption was significantly higher (significant risk factor for carbapenem-resistant A. baumannii acquisition) than other risk factors (P = 0.0123), whereas older age of patients was not significantly higher among other ranges in multivariate analysis by analysis of variance (ANOVA test). The bla(OXA-51-like) gene was the predominant gene, followed by bla(OXA-23-like), bla(OXA-24-like), bla(OXA-58-like), bla(SIM), bla(NDM) and bla(VIM) genes being 66, 35.33, 22, 14, 1.33, 1.33 and 13.33%, respectively. Furthermore, the co-existence of bla(OXA-51-like)/bla(OXA-23-like), bla(OXA-51-like)/bla(OXA-23-like)/bla(OXA-24-like) and bla(OXA-51-like)/bla(OXA-24-like) were 22.67, 12 and 11.33%, respectively. Conclusion: A high level of class D and class B carbapenemases among A. baumannii strains in the burn settings is a crisis in the eradication of infections caused by MDR, XDR and PDR strains. Therefore, the uncontrolled consumption of last-line antibiotics should be restricted and infection control strategies must be implemented accurately. Copyright (C) 2019 Wolters Kluwer Health, Inc. All rights reserved.

High-Quality Complete Genome Sequences of Three Pseudomonas aeruginosa Isolates Retrieved from Patients Hospitalized in Intensive Care Units.

Pseudomonas aeruginosa is one of the major Gram-negative pathogens responsible for hospital-acquired infections. Here, we present high-quality genome sequences of isolates from three P. aeruginosa genotypes retrieved from patients hospitalized in intensive care units. PacBio reads were assembled into a single contig, which was afterward corrected using Illumina HiSeq reads.

Protective and therapeutic application of the depolymerase derived from a novel KN1 genotype of Klebsiella pneumoniae bacteriophage in mice

Klebsiella pneumoniae is one of the major Gram-negative bacterial pathogens causing hospital-acquired multidrug-resistant infections, and the antimicrobial treatment options are scarce. The lack of available antimicrobials has prompted the development of alternative strategies for the treatment of these infections. In this study, a K. pneumoniae bacteriophage (vB_KpnP_IME321) targeting a KN1 capsular type strain, Kp409, was isolated, characterized and sequenced. This bacteriophage has a latent period of 20 min and a burst size of approximately 410 pfu/cell. It contained 49 predicted open reading frames, of which ORF42 was identified as encoding the putative capsule depolymerase. The enzyme expressed and purified in the Escherichia coli BL21 system, namely Dp42, could depolymerize the capsular polysaccharide of Kp409 and form translucent halos on the plates. The phage-encoded depolymerase could increase the inhibitory effect of serum on the growth of bacteria in vitro. Pre-treated with Dp42 rescued 100% of mice following lethal Kp409 challenge, and administration of this enzyme after infection significantly increased survival rates of infected mice in the animal experiment. In conclusion, the phage-encoded depolymerase Dp42 represents a potential alternative strategy for controlling infections mediated by K. pneumoniae expressing the KN1 capsular polysaccharide.

2149. Real-Time Nationwide Surveillance for Antimicrobial Resistance of Major Pathogens Using Automated Data Collection System in Korea: A KARS-Net Study

BackgroundInformation on the most current status of antimicrobial resistance (AMR) in local and national levels has critical importance. However, collection and analysis of a large number of antimicrobial susceptibility test (AST) results often results in additional workload in healthcare facilities and latency in final reporting. We sought to develop an automated nationwide surveillance network in Korea.MethodsData collection servers were set up at each participating institutions, which collects AST results of every bacterial isolate from blood, cerebrospinal fluids, urine, and respiratory specimens. Collected results are anonymized and transmitted to central data server every day without human input. End-user can perform various analyses using data warehouse server through web interface. Only first isolates of same species from individual patients were included in analysis.ResultsA total of 19 hospitals located in various regions in Korea participated to the network. From January 2015 through December 2017, AST results of 347,356 isolates were collected. The proportion of MRSA among S. aureus (n = 17,761) was 65.3%, which declined gradually from 71.5 to 62.3% during study period (P < 0.001). The proportion of VRE increased from 29.3 to 36.3% (P = 0.001). Resistance rates of E. coli (n = 63,628) to third and fourth generation cephalosporins, fluoroquinolone, and piperacillin–tazobactam were 31.6, 23.0, 44.0, and 4.2%, respectively. Resistance rates of K. pneumoniae (n = 16,875) to same classes were 32.2, 28.1, 31.0 and 19.1%, respectively. Among E. coli and K. pneumoniae, 0.4 and 4.3% were resistant to carbapenem. Resistance rates of P. aeruginosa (n = 12,895) to carbapenem was 30.5%. However, 72.7% of A. baumannii isolates (n = 9,885) were resistant to carbapenem. Colistin resistance rate was still low at 0.5%.ConclusionWe have established a fully automated nationwide surveillance network for AMR in Korea. Our system provided data on the most current status of AMR, which revealed increase in resistance rates among major Gram-negative pathogens compared with previous studies.Figure 1.Schematic diagram of the Korean Antimicrobial Resistance Surveillance Network (KARSNet).Figure 2.Temporal trends of the resistance rates of S. aureus and E. faecium.Figure 3.Temporal trends of the resistance rates of major Gram-negative species.Disclosures All authors: No reported disclosures.

The novel metallo-β-lactamase PNGM-1 from a deep-sea sediment metagenome: crystallization and X-ray crystallographic analysis.

Metallo-β-lactamases (MBLs) are present in major Gram-negative pathogens and environmental species, and pose great health risks because of their ability to hydrolyze the β-lactam rings of antibiotics such as carbapenems. PNGM-1 was the first reported case of a subclass B3 MBL protein that was identified from a metagenomic library from deep-sea sediments that predate the antibiotic era. In this study, PNGM-1 was overexpressed, purified and crystallized. Crystals of native and selenomethionine-substituted PNGM-1 diffracted to 2.10 and 2.30 Å resolution, respectively. Both the native and the selenomethionine-labelled PNGM-1 crystals belonged to the monoclinic space group P21, with unit-cell parameters a = 122, b = 83, c = 163 Å, β = 110°. Matthews coefficient (VM) calculations suggested the presence of 6-10 molecules in the asymmetric unit, corresponding to a solvent content of ∼31-58%. Structure determination is currently in progress.

Preliminary Survey of Gram-Negative Bacterial Pathogens from Commonly Caught Fish Species (Oreochromis niloticus, Cyprinus carpio and Clarias gariepinus) in Lake Hayiq, Ethiopia

A cross-sectional study was conducted from October 2016 to May 2017 on Oreochromis niloticus, Cyprinus carpio and Clarias gariepinus fish species at Lake Hayiq, North East Ethiopia with the objective of isolation and identification of major gram-negative bacterial pathogens of commonly catched fish species from Lake Hayik. A total of 98 live fishes (49 Oreochromis niloticus, 30 Cyprinus carpio and 19 Clarias gariepinus) were collected and transported to the laboratory. From the three fish species a total of 384 organ samples (96 skins, 98 gills, 94 intestines, 33 swim bladders, 19 kidneys, and 44 livers) were taken. A systematic random sampling technique was used to select the fish samples after drawn from the water. Among 384 fish organ samples 116 (30.2%) gram negative bacterial isolates were found: 9 (2.3%) Aeromonas species, 24 (6.3%) Pseudomonas species, 6 (1.6%) Enterobacter species, 29 (7.6%) Escherichia species, 4 (1.6%) Edwardsiella species, 11 (2.9%) Klebsiella species, 5 (1.3%) Proteus species, 8 (2.1%) Vibrio species, 14 (3.6%) Flavobacter species and 6 (1.6%) Salmonella species were found. Isolation among organs: Pseudomonas species, skin (33.3%), intestine (33.3%), and swim bladder (12.5%); Escherichia species: intestine (41.4%), skin (24.2%) and gill (24.2%) and Klebsiella species: liver (27.3%), gills (36.4%) were found. All the isolated bacterial species were gram-negative bacteria. From the three fish species, Oreochromis niloticus was the most affected fish species (48.2%) while Clarias gariepinus species were the least affected species (17.2%). In conclusion, majorities of those pathogens isolated and identified were very important for different fish disease outbreaks and also public health importance. But, very few and disintegrated studies with scanty data have been done whereas, it has been nowadays fishery is one of the main growth transformation plan for food security in Ethiopia. Therefore, it needs further integrated investigation on fish bacterial diseases.

Multiplex identification of sepsis-causing Gram-negative pathogens from the plasma of infected blood.

Early and accurate detection of bacterial pathogens in the blood is the most crucial step for sepsis management. Gram-negative bacteria are the most common organisms causing severe sepsis and responsible for high morbidity and mortality. We aimed to develop a method for rapid multiplex identification of clinically important Gram-negative pathogens and also validated whether our system can identify Gram-negative pathogens with the cell-free plasm DNA from infected blood. We designed five MLPA probe sets targeting the genes specific to major Gram-negative pathogens (uidA and lacY for E. coli, ompA for A. baumannii, phoE for K. pneumoniae, and ecfX for P. aeruginosa) and one set targeting the CTX-M group 1 to identify the ESBL producing Gram-negative pathogens. All six target-specific peaks were clearly separated without any non-specific peaks in a multiplex reaction condition. The minimum detection limit was 100 fg of pathogen DNA. When we tested 28 Gram-negative clinical isolates, all of them were successfully identified without any non-specific peaks. To evaluate the clinical applicability, we tested seven blood samples from febrile patients. Three blood culture positive cases showed E. coli specific peaks, while no peak was detected in the other four culture negative samples. This technology can be useful for detection of major sepsis-causing, drug-resistant Gram-negative pathogens and also the major ESBL producing Gram-negatives from the blood of sepsis patients in a clinical setting. This system can help early initiation of effective antimicrobial treatment against Gram-negative pathogens for sepsis patients, which is very crucial for better treatment outcomes.