Isolation and Identification of a Siphoviridae Bacteriophage Targeting Multidrug-resistant Klebsiella pneumoniae

Background: Based on the WHO, multidrug-resistant Klebsiella pneumoniae is a priority pathogen that causes opportunistic infections and is widely spread in the environment. Phage therapy is considered a natural, safe, and very efficient alternative to treat difficult-to-treat infections. Objectives: This study aimed to isolate highly virulent, lytic bacteriophages and evaluate their efficacy for lysing multidrug-resistant K. pneumoniae. Methods: Municipal wastewater samples were collected and filtered using 0.22 µm syringe filters and cultivated with log-phase cultures of K. pneumoniae using enrichment media. After 48 h of incubation, the cultures were centrifuged, and the resultant supernatant was filtered (0.22 µm). The detection of the phage was done using the spot assay with K. pneumoniae as the host. One-step growth kinetics and bacterial reduction tests were conducted to assess the growth kinetics of the isolated phage. The stability of the isolated phage was characterized by subjecting it to various temperature and pH conditions. The chemical stability of the K. pneumoniae phage was determined by exposing it to various organic compounds. A panel of 20 bacterial strains was tested using the spot assay, as well as double agar overlying assay, to determine the host range of the isolated phage. Results: Out of 40 wastewater samples tested, only one sample was tested positive for the K. pneumoniae phage (2.5%) that was lytic against the host strain. The K. pneumoniae phage had a latent period of 15 min and a burst size of 100 virions per infected cell. It was most stable at 37°C and pH range of 6.0 to 10.0. Chemically, the K. pneumoniae phage was resistant to 10% chloroform treatment. Transmission electron micrograph indicated that the K. pneumoniae phage belonged to the order Caudovirales, family Siphoviridae, morphotype B1. Conclusions: Most of the characteristic features of the K. pneumoniae phage indicated the potential of this phage to be used in phage therapy. Hence, a comprehensive study is highly recommended to characterize the K. pneumoniae phage genome, detect its molecular interactions with the host cell, and determine its lytic activity in combination with other phages, which may lead to the efficient utilization of this phage in phage therapy against K. pneumoniae infections.

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Prevalence of Antibiotic-Resistant Bacteria ESKAPE among Healthy People Estimated by Monitoring of Municipal Wastewater

Antibiotics ◽

10.3390/antibiotics10050495 ◽

2021 ◽

Vol 10 (5) ◽

pp. 495

Author(s):

Masateru Nishiyama ◽

Susan Praise ◽

Keiichi Tsurumaki ◽

Hiroaki Baba ◽

Hajime Kanamori ◽

...

Keyword(s):

Antibiotic Resistance ◽

General Population ◽

Municipal Wastewater ◽

Treatment Plant ◽

Multidrug Resistant ◽

Resistant Bacteria ◽

Municipal Wastewater Treatment ◽

Antibiotic Resistant ◽

Carbapenem Resistant ◽

Wastewater Samples

There is increasing attention toward factors that potentially contribute to antibiotic resistance (AR), as well as an interest in exploring the emergence and occurrence of antibiotic resistance bacteria (ARB). We monitored six ARBs that cause hospital outbreaks in wastewater influent to highlight the presence of these ARBs in the general population. We analyzed wastewater samples from a municipal wastewater treatment plant (MWWTP) and hospital wastewater (HW) for six species of ARB: Carbapenem-resistant Enterobacteria (CARBA), extended-spectrum β-lactamase producing Enterobacteria (ESBL), multidrug-resistant Acinetobacter (MDRA), multidrug-resistant Pseudomonas aeruginosa (MDRP), methicillin-resistant Staphylococcus aureus (MRSA), and vancomycin-resistant Enterococci (VRE). We registered a high percentage of ARBs in MWWTP samples (>66%) for all ARBs except for MDRP, indicating a high prevalence in the population. Percentages in HW samples were low (<78%), and no VRE was detected throughout the study. CARBA and ESBL were detected in all wastewater samples, whereas MDRA and MRSA had a high abundance. This result demonstrated the functionality of using raw wastewater at MWWTP to monitor the presence and extent of ARB in healthy populations. This kind of surveillance will contribute to strengthening the efforts toward reducing ARBs through the detection of ARBs to which the general population is exposed.

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Transcriptional Response of Multidrug-Resistant Klebsiella pneumoniae Clinical Isolates to Ciprofloxacin Stress

Canadian Journal of Infectious Diseases and Medical Microbiology ◽

10.1155/2021/5570963 ◽

2021 ◽

Vol 2021 ◽

pp. 1-6

Author(s):

Roman Farooq Alvi ◽

Bilal Aslam ◽

Muhammad Hidayat Rasool ◽

Saima Muzammil ◽

Abu Baker Siddique ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Stress Responses ◽

Genetic Resistance ◽

Transcriptional Response ◽

Multidrug Resistant ◽

Mrna Levels ◽

Dependent Manner ◽

Isolation And Identification ◽

Concentration Dependent Manner ◽

High Level

Background. The term “persisters” refers to a small bacterial population that persists during treatment with high antibiotic concentration or dose in the absence of genetic resistance. The present study was designed to investigate the transcriptional response in indigenous Klebsiella pneumoniae under the ciprofloxacin stress. Methods. Isolation and identification of K. pneumoniae were carried out through standard microbiological protocols. The characterization of quinolone resistance was performed by estimating the quinolone susceptibility testing, MIC estimation, and detecting the QRDR and PMQR. Transcriptional response of the isolates to ciprofloxacin was determined using qPCR. Results. Among 34 isolates, 23 (67%) were resistant to ciprofloxacin. Both QRDR (gyrA and gyrB) and PMQR (qnrA, qnrB, and qnrS) were detected in the isolates, and all were found resistant to ciprofloxacin. The mRNA levels of both mutS and euTu under the influence of ciprofloxacin were significantly increased. On ciprofloxacin exposure, the mRNA levels of the DNA damage response element (mutS) were raised in a time-dependent fashion. K. pneumoniae showed high-level resistance to ciprofloxacin in the presence of mutations in QRDR and PMQR genes. Conclusion. The transcriptional response revealed the upregulation of DNA repair and protein folding elements (mutS and euTu) in ciprofloxacin stress and delayed cell division. The ciprofloxacin was found to trigger various stress responses in a time- and concentration-dependent manner.

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The Isolation of specifically lytic phages along with their extracted endolysins as antibacterial agents to MDR Enterococcus faecalis

Research Journal of Pharmacy and Technology ◽

10.52711/0974-360x.2021.00791 ◽

2021 ◽

pp. 4547-4554

Author(s):

Shaymaa Husham Ahmed ◽

Rand R. Hafidh

Keyword(s):

Enterococcus Faecalis ◽

Bacterial Growth ◽

Phage Therapy ◽

Burst Size ◽

Alternative Therapy ◽

Multidrug Resistant ◽

Multiple Drug ◽

Specific Phage ◽

Multiple Drug Resistant ◽

Phage Cocktail

Background: Multidrug-resistant (MDR) enterococci have become a major problem in recent times and have been reported increasingly around the world. Lytic phages infect bacteria leading to rapid host death with limited risk of phage transduction, underlining the increasing interest in potential phage therapy in the future. Objective (s): The aim of this study is to use phage therapy as alternative approach for treatment of Enterococcus faecalis infections that recorded as MDR in Iraq to tackle this problem. Materials and Methods: Thirty E. faecalis isolates were collected from patients with different infectious diseases such as urinary tract infection (UTI), diabetic foot, septicemia, and wound infections. The isolation of specific lytic phages was from different environmental sources such as (sewage, and wastewater). The biokinetic assays were carried out to measure the characteristics of the isolated phage. The study of the bacteriophage and the formed phage cocktail infectivity against isolates E.faecalis was tested by the top layer assay. The phage endolysin was extracted from the best bacteriophage that gave best results. Results: All the isolated E.faecalis was reported as MDR in this study. About 75 E.faecalis specific phages were isolated and purified. All the isolated bacteria were 100% sensitive to the lytic phages. The formed phage cocktail was capable to create inhibition zones on the most bacterial isolates' lawns. The molecular weight and the concentration of the extracted endolysin was evaluated in this study and found to be as (48 kDa) and (0.5mg/ml), respectively. The antibacterial activity of the extracted endolysin was evaluated by the turbidity reduction assay. A clear decline in the bacterial growth was manifested (5x107 CFU/ml) to (1x104 CFU/ml), in which the bacterial growth was reduced by (3.63 log). The endolysin found to be effective against 90% of E.faeclais isolates. Conclusion: The activity of the isolated specific phage together with the activity of the formed phage cocktail, were efficient as successful and inexpensive method of therapy against MDR E.faecalis. The potential of the extracted endolysin over the phage therapy was verified in this study. The coverage rate and the absence of resistant E.faecalis to the phage and its endolysin had emphasized on the importance of this alternative therapy to commonly used antibiotics. List of abbreviation: MDR = multiple drug-resistant, E. faecalis = Enterococcus faecalis, BT = burst time, BS = burst size, IP = infective percentage, Phage =bacteriophage, CFU = Colony forming unit, kDa = Kilodalton, OD = Optical Densities.

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Isolation and characterization of lytic phage TUN1 specific for Klebsiella pneumoniae K64 clinical isolates from Tunisia

BMC Microbiology ◽

10.1186/s12866-021-02251-w ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Simone Eckstein ◽

Jana Stender ◽

Sonia Mzoughi ◽

Kilian Vogele ◽

Jana Kühn ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Antibiotic Therapy ◽

Geographical Location ◽

Case Fatality ◽

Multidrug Resistant ◽

Military Hospital ◽

Lytic Phage ◽

Isolation And Characterization ◽

Wastewater Samples

Abstract Background Multidrug-resistant Klebsiella pneumoniae spp. (kp) are emerging agents of severe infections of the respiratory, urinary tract and wounds that can progress to fatal septicemia. The use of bacteriophages is currently being considered as an effective alternative or adjuvant to antibiotic therapy. Results In this study, we report capsule (K)-typing of 163 carbapenem-resistant Kp (CRKP) isolated 2014–2018 at the Military Hospital of Instruction of Tunis (MHT), Tunisia, by partial amplification and sequencing of the Kp wzi gene. The most prevalent K-type overall was K64 with 50.3% followed by K17 and K27 (22.7 and 11.0%, respectively). K64 Kp strains were most common and associated with increased case/fatality rates, especially at the intensive care unit (ICU). Using a K64 Kp strain we isolated and characterized a lytic Kp phage, vB_KpP_TUN1 (phage TUN1), from wastewater samples of the ICU at the MHT. TUN1 belongs to the Autographiviridae family and specifically digests K64 Kp capsules most probably via a depolymerase encoded by gp47. Furthermore, we successfully assembled phage TUN1 in a non-replicative host (E. coli) raising the possibility of in vitro assembly in the absence of live bacterial hosts. We propose that phage TUN1 is a promising candidate to be used as an adjuvant or an alternative to antibiotic therapy in CRKP infections, facilitating regulatory approval of phage therapy. Conclusions K64, K17 and K27 are the most common wzi capsule types in this geographical location in Northern Africa. The lytic phage TUN1 efficiently lyses K64 Kp strains associated with increased case/fatality rates at body temperature. Together with its ability to be rescued in a non-replicative host these features enhance the utility of this phage as an antibacterial agent.

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Characterization and genome analysis of novel phage vB_KpnM _Bp5 infecting Klebsiella pneumonia

10.21203/rs.2.13213/v1 ◽

2019 ◽

Author(s):

Cong Zhang ◽

Chenling Ge ◽

Xiaoye Wang ◽

Deyuan Wei ◽

Xun Li ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Genome Analysis ◽

Bacterial Resistance ◽

Burst Size ◽

Multidrug Resistant ◽

Lytic Phage ◽

Klebsiella Pneumonia ◽

Large Outbreak ◽

Electron Microscopy Analysis ◽

Blastn Analysis

Abstract Background With the incidence of antibiotic resistance reaching crisis point, it is imperative to find alternative treatments for multidrug-resistant infections. Using phage for pathogen control might be a promising treatment option to combat bacterial resistance. Results In this study, a lytic phage, designated vB_KpnM _Bp5, was isolated from pig faecal sample in Nanning, Guangxi province of China, and classified as a member of the family Muscle virus based on electron microscopy analysis. A one-step growth curve of the phage at the optimal MOI revealed that the latent time was 40 min and the burst size was 24 PFU/cell, indicative of good lysis capacity. Whole genome sequencing showed that phage vB_KpnM _Bp5 had a small dsDNA genome of 43872 bp. BLASTn analysis showed that it shared 94.06% identity (94% genome coverage) with Klebsiella phage vB_KpnP_SU552A of complete genome idefix. RAST genome analysis showed that the phage had 50 ORFs due to its small genome size, and the number of functional proteins was consistent with other phages. To evaluate the therapeutic effect of Klebsiella pneumoniae infection in mice, the results showed that phages provided vB_KpnM _Bp5better protection. Conclusion The phage vB_KpnM _Bp5 had the characteristics of broad host spectrum, strong environmental adaptability, short incubation period, large outbreak amount, and can cure the mouse model infected by Klebsiella pneumoniae. These findings suggested that phage vB_KpnM _Bp5 could be considered a potential therapeutic or prophylactic candidate against Klebsiella pneumonia infection.

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New Insight on Antibiotic Resistance and Virulence of Escherichia coli from Municipal and Animal Wastewater

Antibiotics ◽

10.3390/antibiotics10091111 ◽

2021 ◽

Vol 10 (9) ◽

pp. 1111

Author(s):

Gabriela Gregova ◽

Vladimir Kmet ◽

Tatiana Szaboova

Keyword(s):

Escherichia Coli ◽

Antibiotic Resistance ◽

Municipal Wastewater ◽

Wastewater Treatment Plants ◽

Multidrug Resistant ◽

Wastewater Sample ◽

E Coli ◽

Animal Wastewater ◽

Wastewater Samples ◽

One Year

Antibiotic resistance of the indicator microorganism Escherichia coli was investigated in isolates from samples collected during the course of one year from two wastewater treatment plants treating municipal and animal wastes in Slovakia, respectively. The genes of antibiotic resistance and virulence factors in selected resistant E. coli isolates were described. A high percentage of the isolates from municipal and animal wastewater were resistant to ampicillin, streptomycin, tetracycline, ceftiofur, ceftriaxone, and enrofloxacin. In the selected E. coli isolates, we detected the following phenotypes: ESBL (20.4% in animal wastewater; 7.7% in municipal wastewater), multidrug-resistant (17% of animal and 32% of municipal isolates), high resistance to quinolones (25% of animal and 48% of municipal samples), and CTX-M (7.9% of animal and 17.3% of municipal isolates). We confirmed an integro-mediated antibiotic resistance in 13 E. coli strains from municipal and animal wastewater samples, of which the Tn3 gene and virulence genes cvaC, iutA, iss, ibeA, kps, and papC were detected in six isolates. One of the strains of pathogenic E. coli from the animal wastewater contained genes ibeA with papC, iss, kpsII, Int1, Tn3, and Cit. In addition, one blaIMP gene was found in the municipal wastewater sample. This emphasises the importance of using the appropriate treatment methods to reduce the counts of antibiotic-resistant microorganisms in wastewater effluent.

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Bacteriophage Treatment Rescues Mice Infected with Multidrug-Resistant Klebsiella pneumoniae ST258

mBio ◽

10.1128/mbio.00034-21 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Shayla Hesse ◽

Natalia Malachowa ◽

Adeline R. Porter ◽

Brett Freedman ◽

Scott D. Kobayashi ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Alternative Treatment ◽

Bacterial Infections ◽

Phage Therapy ◽

Treatment Approach ◽

Multidrug Resistant ◽

Sequence Type ◽

Infection Model ◽

Content Type ◽

Treatment Groups

ABSTRACT Severe infections caused by multidrug-resistant Klebsiella pneumoniae sequence type 258 (ST258) highlight the need for new therapeutics with activity against this pathogen. Bacteriophage (phage) therapy is an alternative treatment approach for multidrug-resistant bacterial infections that has shown efficacy in experimental animal models and promise in clinical case reports. In this study, we assessed microbiologic, histopathologic, and survival outcomes following systemic administration of phage in ST258-infected mice. We found that prompt treatment with two phages, either individually or in combination, rescued mice with K. pneumoniae ST258 bacteremia. Among the three treatment groups, mice that received combination phage therapy demonstrated the greatest increase in survival and the lowest frequency of phage resistance among bacteria recovered from mouse blood and tissue. Our findings support the utility of phage therapy as an approach for refractory ST258 infections and underscore the potential of this treatment modality to be enhanced through strategic phage selection. IMPORTANCE Infections caused by multidrug-resistant K. pneumoniae pose a serious threat to at-risk patients and present a therapeutic challenge for clinicians. Bacteriophage (phage) therapy is an alternative treatment approach that has been associated with positive clinical outcomes when administered experimentally to patients with refractory bacterial infections. Inasmuch as these experimental treatments are prepared for individual patients and authorized for compassionate use only, they lack the rigor of a clinical trial and therefore cannot provide proof of efficacy. Here, we demonstrate that administration of viable phage provides effective treatment for multidrug-resistant K. pneumoniae (sequence type 258 [ST258]) bacteremia in a murine infection model. Moreover, we compare outcomes among three distinct phage treatment groups and identify potential correlates of therapeutic phage efficacy. These findings constitute an important first step toward optimizing and assessing phage therapy’s potential for the treatment of severe ST258 infection in humans.

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Detection and prevalence of multidrug-resistant Klebsiella pneumoniae strains isolated from poultry farms in Blitar, Indonesia

Biodiversitas Journal of Biological Diversity ◽

10.13057/biodiv/d211024 ◽

2020 ◽

Vol 21 (10) ◽

Author(s):

DIAN AYU PERMATASARI ◽

ADIANA MUTAMSARI WITANINGRUM ◽

FRESHINTA JELLIA WIBISONO ◽

Mustofa Helmi Effendi

Keyword(s):

Klebsiella Pneumoniae ◽

Antimicrobial Agents ◽

Multidrug Resistant ◽

Multi Drug Resistance ◽

Resistant Bacteria ◽

Isolation And Identification ◽

Layer Chicken ◽

Confirmation Test ◽

Poultry Farms ◽

The Impact

Abstract. Permatasari DA, Witaningrum AM, Wibisono FJ, Effendi MH. 2020. Detection and prevalence of multidrug-resistant Klebsiella pneumoniae strain isolated from poultry farms in Blitar, Indonesia. Biodiversitas 21: 4642-4647. Antibiotics are commonly used as therapy and disease control in humans and animals. However, the widespread use of antibiotics may also trigger the rise of antibiotic resistance. Therefore, the aim of this study was to study the occurrence of Multidrug-Resistant (MDR) of Klebsiella pneumoniae from poultry farms in Blitar. Chicken cloacal swabs were collected from 63 poultry farms (32 broilers and 31 layers) were taken randomly, using total samples 160 broilers and 155 layers. The collected swab samples were inoculated on MacConkey agar medium for isolation and identification. Single colonies were isolated after primary positive cultures and identified by using the IMViC test and TSIA. 28 (8.88%) out of samples were positive for Klebsiella pneumoniae. The antimicrobial confirmation test showed that 53.57% of the K. pneumoniae isolates were Multi-Drug Resistance (MDR) bacteria. The percentage of MDR bacteria against different antibiotics included ampicillin 75%, Erythromycin 42.86%, Tetracycline 35.72%, Sulfamethoxazole 32.14% and Streptomycin 21.4%. This study confirms that the percentage of resistant isolates of Klebsiella pneumoniae from 5 antimicrobial agents of broiler chicken is higher than layer chicken. The presence of multidrug-resistant bacteria is a threat to public health and livestock. The impact of these conditions leaves a limited treatment option as chicken farmers in Indonesia still using antibiotics without veterinarian supervision.

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Phenotypic Characterization of Extended Spectrum Beta-lactamase, Class C Cephalosporinase and Carbapenemase-Producing Klebsiella Species Isolated from Patients Consulted at Four Yaounde-Based Hospitals.

10.21203/rs.3.rs-793227/v1 ◽

2021 ◽

Author(s):

Emilia Enjema Lyonga Mbamyah ◽

Mangum Patience Kumcho ◽

Michel Toukam ◽

Dieudonné Sedena ◽

Florence Anjabie Enyeji ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Opportunistic Infections ◽

Acquired Resistance ◽

Multidrug Resistant ◽

Phenotypic Characterization ◽

Beta Lactamase ◽

Klebsiella Species ◽

Extended Spectrum Beta Lactamase ◽

Extended Spectrum ◽

Class C

Abstract Background: Klebsiella spp. are bacteria of medical importance for their role in opportunistic infections. These infections are often difficult to treat because of acquired resistance to one or several families of antimicrobials. The present study aimed at detecting Extended Spectrum Beta-lactamase (ESBL), Class C cephalosporinase (AmpC) and carbapenemase resistant phenotypes of Klebsiella spp. isolated from patients consulted at four Yaounde-based hospitals. Results: The frequency of the species isolated was Klebsiella pneumoniae (69%), K. oxytoca (14%), K. ozaenae (12%) and K. rhinoscleromatis (5%). Isolates were most resistant to penicillins (90%), sulphonamides (84%), cepaholosporins (80%), and least resistant to carbapenems (10.2%). Three isolates namely: two K. oxytoca and one K. pneumoniae were resistant to all twenty-eight (28) antibiotics tested. Klebsiella pneumoniae was the species with the most multidrug resistant isolates (59.4%). Most isolates (83.6%) expressed at least one resistance phenotype, while 63.6% of the isolates expressed all three phenotypes. Many of the isolates were ESBL producers (71.6%), while fewer isolates were carbapenemase (26.7%) and AmpC (6.6%) producers. Three carbapenemases (Klebsiella pneumoniae carbapenemase-KPC, Metallo-Beta Lactamase-MBL and OXA-48) were detected from 26.7% of the isolates and the combination KPC and MBL were the most detected phenotypes (12.9%). Conclusion: These results reveal that resistance of Klebsiella spp. to cephalosporins is high and this may be exacerbated as a result of the co-expression of AmpC and carbapenemases. About a quarter of the isolates had acquired carbapenemases that confer resistance to all beta-lactamases and carbapenems which constitute last line drugs. The resistance burden is further strengthened in isolates that acquired more than one carbapenemase aggravating associated patient morbidity and mortality. Therefore, it is necessary to continue monitoring antimicrobial resistance of local strains for better informed decisions on empirical treatment guide and better patient care.

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Complete Genome Sequence of a Novel Multidrug-Resistant Klebsiella pneumoniae Phage, vB_Kpn_IME260

Genome Announcements ◽

10.1128/genomea.00055-17 ◽

2017 ◽

Vol 5 (19) ◽

Author(s):

Shaozhen Xing ◽

Xiangchun Pan ◽

Qiang Sun ◽

Guangqian Pei ◽

Xiaoping An ◽

...

Keyword(s):

Public Health ◽

Klebsiella Pneumoniae ◽

Phage Therapy ◽

Bacterial Pathogen ◽

Genomic Data ◽

Multidrug Resistant ◽

Resistant Bacteria ◽

Drug Resistant ◽

Content Type ◽

Drug Resistant Bacteria

ABSTRACT Klebsiella pneumoniae is the most common clinically important opportunistic bacterial pathogen and its infection is often iatrogenic. Its drug resistance poses a grave threat to public health. The genomic data reported here comprise an important resource for research on phage therapy in the control of drug-resistant bacteria.

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