Phenotypic and molecular evaluation of biofilm formation in Klebsiella pneumoniae carbapenemase (KPC) isolates obtained from a hospital of Pelotas, RS, Brazil

2021 ◽  
Vol 70 (11) ◽  
Author(s):  
Letícia Roloff Stallbaum ◽  
Beatriz Bohns Pruski ◽  
Suelen Cavalheiro Amaral ◽  
Stella Buchhorn de Freitas ◽  
Daniela Rodriguero Wozeak ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Klebsiella Pneumoniae ◽  
Crystal Violet ◽  
Biofilm Formation ◽  
Staining Method ◽  
Multidrug Resistant ◽  
Crystal Violet Staining ◽  
Content Type ◽  
Klebsiella Pneumoniae Carbapenemase

Introduction. A significant cause of mortality in the intensive care unit (ICU) is multidrug-resistant (MDR) Gram-negative bacteria, such as Klebsiella pneumoniae carbapenemase (KPC). Biofilm production is a key factor in KPC colonization and persistence in the host, making the treatment difficult. Gap Statement. The aim of this study was to evaluate the antibiotic resistance, molecular and phenotypic biofilm profiles of 12 KPC isolates associated with nosocomial infection in a hospital in Pelotas, Rio Grande do Sul, Brazil. Methodology. Clinical isolates were obtained from different sources, identified and characterized by antibiotic resistance and carbapenemase synthesis following the Clinical and Laboratory Standards Institute (CLSI) guidelines. Polymerase chain reaction (PCR) was used to evaluate the presence of carbapenemase (blaKPC ) and biofilm formation-associated genes (fimA, fimH, rmpA, ecpA, mrkD and wabG). Additionally, phenotypic evaluation of in vitro biofilm formation capacity was evaluated by Congo red agar (CRA) assay and the crystal violet staining method. Results. The 12 isolates evaluated in this study presented the blaKPC gene and were positive for synthesizing carbapenemases in vitro. In the carbapenem class, 83.3 % isolates were resistant and 16.7 % intermediately resistant to imipenem and meropenem. Molecular analyses found that the fimA and wabG genes were detected in 75 % of isolates, while fimH and ecpA were detected in 42 % and mrkD were detected in 8.3 % (1). The CRA assay demonstrated that all isolates were slime producers and 91.7 % (11) of isolates were classified as strong and 8.3 % (1) as moderate biofilm producers by the crystal violet staining method. The optical density (OD540nm) for strong biofilm formers ranged from 0.80±0.05 to 2.47±0.28 and was 0.55±0.12 for moderate biofilm formers. Conclusion. Our study revealed a high level of antibiotic resistance and biofilm formation in KPC isolates obtained from a hospital in Pelotas, RS, Brazil.

scholarly journals In Vitro Study of Antimicrobial Percutaneous Nephrostomy Catheters for Prevention of Renal Infections

2017 ◽  
Vol 61 (6) ◽  
Author(s):  
Nylev Vargas-Cruz ◽  
Ruth A. Reitzel ◽  
Joel Rosenblatt ◽  
Mohamed Jamal ◽  
Ariel D. Szvalb ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Biofilm Formation ◽  
In Vitro Study ◽  
Percutaneous Nephrostomy ◽  
Multidrug Resistant ◽  
Gram Positive ◽  
Gram Negative ◽  
Content Type ◽  
Fungal Organisms

ABSTRACT Percutaneous nephrostomy (PCN) catheters are the primary method for draining ureters obstructed by malignancy and preventing a decline of renal function. However, PCN catheter-related infections, such as pyelonephritis and urosepsis, remain a significant concern. Currently, no antimicrobial PCN catheters are available for preventing infection complications. Vascular catheters impregnated with minocycline-rifampin (M/R) and M/R with chlorhexidine coating (M/R plus CHD) have previously demonstrated antimicrobial activity. Therefore, in this study, we examined whether these combinations could be applied to PCN catheters and effectively inhibit biofilm formation by common uropathogens. An in vitro biofilm colonization model was used to assess the antimicrobial efficacy of M/R and M/R-plus-CHD PCN catheters against nine common multidrug-resistant Gram-positive and Gram-negative uropathogens as well as Candida glabrata and Candida albicans. Experimental catheters were also assessed for durability of antimicrobial activity for up 3 weeks. PCN catheters coated with M/R plus CHD completely inhibited biofilm formation for up to 3 weeks for all the organisms tested. The reduction in colonization compared to uncoated PCN catheters was significant for all Gram-positive, Gram-negative, and fungal organisms (P < 0.05). M/R-plus-CHD PCN catheters also produced significant reductions in biofilm colonization relative to M/R PCN catheters for Enterobacter spp., Escherichia coli, Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci, C. glabrata, and C. albicans (P < 0.05). M/R-plus-CHD PCN catheters proved to be highly efficacious in preventing biofilm colonization when exposed to multidrug-resistant pathogens common in PCN catheter-associated pyelonephritis. M/R-plus-CHD PCN catheters warrant evaluation in a clinical setting to assess their ability to prevent clinically relevant nephrostomy infections.

scholarly journals Antibody-Based Immunotherapy To Treat and Prevent Infection with Hypervirulent Klebsiella pneumoniae

2016 ◽  
Vol 24 (1) ◽  
Author(s):  
Elizabeth Diago-Navarro ◽  
Isabel Calatayud-Baselga ◽  
Donglei Sun ◽  
Camille Khairallah ◽  
Inderjit Mann ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Klebsiella Pneumoniae ◽  
Intravital Microscopy ◽  
Protective Antigen ◽  
Protective Efficacy ◽  
Mesenteric Lymph Nodes ◽  
Content Type ◽  
Anthrax Protective Antigen

ABSTRACT Hypervirulent Klebsiella pneumoniae (hvKp) strains are predicted to become a major threat in Asia if antibiotic resistance continues to spread. Anticapsular antibodies (Abs) were developed because disseminated infections caused by hvKp are associated with significant morbidity and mortality, even with antibiotic-sensitive strains. K1-serotype polysaccharide capsules (K1-CPS) are expressed by the majority of hvKp strains. In this study, K1-CPS-specific IgG Abs were generated by conjugation of K1-CPS to immunogenic anthrax protective antigen (PA) protein. Opsonophagocytic efficacy was measured in vitro and in vivo by intravital microscopy in murine livers. In vivo protection was tested in murine models, including a novel model for dissemination in hvKp-colonized mice. Protective efficacy of monoclonal antibodies (MAbs) 4C5 (IgG1) and 19A10 (IgG3) was demonstrated both in murine sepsis and pulmonary infection. In hvKp-colonized mice, MAb treatment significantly decreased dissemination of hvKp from the gut to mesenteric lymph nodes and organs. Intravital microscopy confirmed efficient opsonophagocytosis and clearance of bacteria from the liver. In vitro studies demonstrate that MAbs work predominantly by promoting FcR-mediated phagocytosis but also indicate that MAbs enhance the release of neutrophil extracellular traps (NETs). In anticipation of increasing antibiotic resistance, we propose further development of these and other Klebsiella-specific MAbs for therapeutic use.

scholarly journals In Vitro Discordance with In Vivo Activity: Humanized Exposures of Ceftazidime-Avibactam, Aztreonam, and Tigecycline Alone and in Combination against New Delhi Metallo-β-Lactamase-Producing Klebsiella pneumoniae in a Murine Lung Infection Model

2017 ◽  
Vol 61 (7) ◽  
Author(s):  
M. L. Monogue ◽  
L. M. Abbo ◽  
R. Rosa ◽  
J. F. Camargo ◽  
O. Martinez ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Lung Infection ◽  
Multidrug Resistant ◽  
Infection Model ◽  
New Delhi ◽  
Beta Lactamase ◽  
Bacterial Reduction ◽  
Content Type

ABSTRACT The management of infections with New Delhi metallo-beta-lactamase-1 (NDM)-producing bacteria remains clinically challenging given the multidrug resistant (MDR) phenotype associated with these bacteria. Despite resistance in vitro, ceftazidime-avibactam previously demonstrated in vivo activity against NDM-positive Enterobacteriaceae. Herein, we observed in vitro synergy with ceftazidime-avibactam and aztreonam against an MDR Klebsiella pneumoniae harboring NDM. In vivo, humanized doses of ceftazidime-avibactam monotherapy resulted in >2 log10 CFU bacterial reduction; therefore, no in vivo synergy was observed.

scholarly journals Identification of Capsular Types in Carbapenem-Resistant Klebsiella pneumoniae Strains bywzcSequencing and Implications for Capsule Depolymerase Treatment

2014 ◽  
Vol 59 (2) ◽  
pp. 1038-1047 ◽  
Author(s):  
Yi-Jiun Pan ◽  
Tzu-Lung Lin ◽  
Yi-Tsung Lin ◽  
Po-An Su ◽  
Chun-Tang Chen ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Vaccine Development ◽  
Survival Rates ◽  
Multidrug Resistant ◽  
Enzyme Treatment ◽  
Capsular Type ◽  
Content Type ◽  
Carbapenem Resistant ◽  
Sequence Types

ABSTRACTKlebsiella pneumoniaeis an important human pathogen associated with a variety of diseases, and the prevalence of multidrug-resistantK. pneumoniae(MDRKP) is rapidly increasing. Here we determined the capsular types of 85 carbapenem-resistantK. pneumoniae(CRKP) strains bywzcsequencing and investigated the presence of carbapenemases and integrons among CRKP strains. Ten CRKP strains (12%) were positive for carbapenemase (imipenemase, 6/85 strains;K. pneumoniaecarbapenemase, 3/85 strains; Verona integron-encoded metallo-β-lactamase, 1/85 strains). Capsular type K64 accounted for 32 CRKP strains (38%), followed by K62 (13%), K24 (8%), KN2 (7%), and K28 (6%). Sequence types (STs) were determined by multilocus sequence typing (MLST), and the results indicated that ST11, which accounted for 47% of these CRKP strains (40/85 strains), was the major ST. We further isolated a K64-specific capsule depolymerase (K64dep), which could enhance serum and neutrophil killingin vitroand increase survival rates for K64K. pneumoniae-inoculated mice. The toxicity study demonstrated that mice treated with K64dep showed normal biochemical parameters and no significant histopathological changes of liver, kidney, and spleen, indicating that enzyme treatment did not cause toxicity in mice. Therefore, the findings of capsular type clustering among CRKP strains and effective treatment with capsule depolymerase for MDRKP infections are important for capsule-based vaccine development and therapy.

scholarly journals Increased Biofilm Formation by Nontypeable Haemophilus influenzae Isolates from Patients with Invasive Disease or Otitis Media versus Strains Recovered from Cases of Respiratory Infections

2014 ◽  
Vol 80 (22) ◽  
pp. 7088-7095 ◽  
Author(s):  
Carmen Puig ◽  
Arnau Domenech ◽  
Junkal Garmendia ◽  
Jeroen D. Langereis ◽  
Pascal Mayer ◽  
...  
Keyword(s):  
Haemophilus Influenzae ◽  
Respiratory Disease ◽  
Otitis Media ◽  
Crystal Violet ◽  
Solid Surface ◽  
Biofilm Formation ◽  
Invasive Disease ◽  
Crystal Violet Staining ◽  
Content Type ◽  
Initial Adhesion

ABSTRACTBiofilm formation by nontypeable (NT)Haemophilus influenzaeremains a controversial topic. Nevertheless, biofilm-like structures have been observed in the middle-ear mucosa of experimental chinchilla models of otitis media (OM). To date, there have been no studies of biofilm formation in large collections of clinical isolates. This study aimed to investigate the initial adhesion to a solid surface and biofilm formation by NTH. influenzaeby comparing isolates from healthy carriers, those with noninvasive respiratory disease, and those with invasive respiratory disease. We used 352 isolates from patients with nonbacteremic community-acquired pneumonia (NB-CAP), chronic obstructive pulmonary disease (COPD), OM, and invasive disease and a group of healthy colonized children. We then determined the speed of initial adhesion to a solid surface by the BioFilm ring test and quantified biofilm formation by crystal violet staining. Isolates from different clinical sources displayed high levels of biofilm formation on a static solid support after growth for 24 h. We observed clear differences in initial attachment and biofilm formation depending on the pathology associated with NTH. influenzaeisolation, with significantly increased biofilm formation for NTH. influenzaeisolates collected from patients with invasive disease and OM compared with NTH. influenzaeisolates from patients with NB-CAP or COPD and healthy colonized subjects. In all cases, biofilm structures were detached by proteinase K treatment, suggesting an important role for proteins in the initial adhesion and static biofilm formation measured by crystal violet staining.

scholarly journals Inhibition of Pseudomonas aeruginosa by Peptide-Conjugated Phosphorodiamidate Morpholino Oligomers

2017 ◽  
Vol 61 (4) ◽  
Author(s):  
James J. Howard ◽  
Carolyn R. Sturge ◽  
Dina A. Moustafa ◽  
Seth M. Daly ◽  
Kimberly R. Marshall-Batty ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antibiotic Resistance ◽  
Polymyxin B ◽  
Multidrug Resistant ◽  
Essential Genes ◽  
Content Type ◽  
Novel Approach ◽  
Phosphorodiamidate Morpholino Oligomers

ABSTRACT Pseudomonas aeruginosa is a highly virulent, multidrug-resistant pathogen that causes significant morbidity and mortality in hospitalized patients and is particularly devastating in patients with cystic fibrosis. Increasing antibiotic resistance coupled with decreasing numbers of antibiotics in the developmental pipeline demands novel antibacterial approaches. Here, we tested peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs), which inhibit translation of complementary mRNA from specific, essential genes in P. aeruginosa. PPMOs targeted to acpP, lpxC, and rpsJ, inhibited P. aeruginosa growth in many clinical strains and activity of PPMOs could be enhanced 2- to 8-fold by the addition of polymyxin B nonapeptide at subinhibitory concentrations. The PPMO targeting acpP was also effective at preventing P. aeruginosa PAO1 biofilm formation and at reducing existing biofilms. Importantly, treatment with various combinations of a PPMO and a traditional antibiotic demonstrated synergistic growth inhibition, the most effective of which was the PPMO targeting rpsJ with tobramycin. Furthermore, treatment of P. aeruginosa PA103-infected mice with PPMOs targeting acpP, lpxC, or rpsJ significantly reduced the bacterial burden in the lungs at 24 h by almost 3 logs. Altogether, this study demonstrates that PPMOs targeting the essential genes acpP, lpxC, or rpsJ in P. aeruginosa are highly effective at inhibiting growth in vitro and in vivo. These data suggest that PPMOs alone or in combination with antibiotics represent a novel approach to addressing the problems associated with rapidly increasing antibiotic resistance in P. aeruginosa.

scholarly journals In Vitro and In Vivo Activities of β-Lactams in Combination with the Novel β-Lactam Enhancers Zidebactam and WCK 5153 against Multidrug-Resistant Metallo-β-Lactamase-Producing Klebsiella pneumoniae

2019 ◽  
Vol 63 (5) ◽  
Author(s):  
Bartolome Moya ◽  
Isabel M. Barcelo ◽  
Gabriel Cabot ◽  
Gabriel Torrens ◽  
Snehal Palwe ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Klebsiella Pneumoniae ◽  
Molecular Targets ◽  
Multidrug Resistant ◽  
The Novel ◽  
Penicillin Binding Protein ◽  
Content Type ◽  
Time Kill

ABSTRACT Zidebactam and WCK 5153 are novel bicyclo-acyl hydrazide (BCH) agents that have previously been shown to act as β-lactam enhancer (BLE) antibiotics in Pseudomonas aeruginosa and Acinetobacter baumannii. The objectives of this work were to identify the molecular targets of these BCHs in Klebsiella pneumoniae and to investigate their potential BLE activity for cefepime and aztreonam against metallo-β-lactamase (MBL)-producing strains in vitro and in vivo. Penicillin binding protein (PBP) binding profiles were determined by Bocillin FL assay, and 50% inhibitory concentrations (IC50s) were determined using ImageQuant TL software. MICs and kill kinetics for zidebactam, WCK 5153, and cefepime or aztreonam, alone and in combination, were determined against clinical K. pneumoniae isolates producing MBLs VIM-1 or NDM-1 (plus ESBLs and class C β-lactamases) to assess the in vitro enhancer effect of BCH compounds in conjunction with β-lactams. Additionally, murine systemic and thigh infection studies were conducted to evaluate BLE effects in vivo. Zidebactam and WCK 5153 showed specific, high PBP2 affinity in K. pneumoniae. The MICs of BLEs were >64 μg/ml for all MBL-producing strains. Time-kill studies showed that a combination of these BLEs with either cefepime or aztreonam provided 1 to >3 log10 kill against MBL-producing K. pneumoniae strains. Furthermore, the bactericidal synergy observed for these BLE–β-lactam combinations translated well into in vivo efficacy even in the absence of MBL inhibition by BLEs, a characteristic feature of the β-lactam enhancer mechanism of action. Zidebactam and WCK 5153 are potent PBP2 inhibitors and display in vitro and in vivo BLE effects against multidrug-resistant (MDR) K. pneumoniae clinical isolates producing MBLs.

scholarly journals Therapeutic Efficacy of Novel Antimicrobial Peptide AA139-Nanomedicines in a Multidrug-Resistant Klebsiella pneumoniae Pneumonia-Septicemia Model in Rats

2020 ◽  
Vol 64 (9) ◽  
Author(s):  
Hessel van der Weide ◽  
Unai Cossío ◽  
Raquel Gracia ◽  
Yvonne M. te Welscher ◽  
Marian T. ten Kate ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Therapeutic Efficacy ◽  
Antimicrobial Agents ◽  
Polymeric Nanoparticles ◽  
Multidrug Resistant ◽  
Antimicrobial Activities ◽  
Bacterial Killing ◽  
Gram Negative ◽  
Content Type

ABSTRACT Antimicrobial peptides (AMPs) have seen limited clinical use as antimicrobial agents, largely due to issues relating to toxicity, short biological half-life, and lack of efficacy against Gram-negative bacteria. However, the development of novel AMP-nanomedicines, i.e., AMPs entrapped in nanoparticles, has the potential to ameliorate these clinical problems. The authors investigated two novel nanomedicines based on AA139, an AMP currently in development for the treatment of multidrug-resistant Gram-negative infections. AA139 was entrapped in polymeric nanoparticles (PNPs) or lipid-core micelles (MCLs). The antimicrobial activity of AA139-PNP and AA139-MCL was determined in vitro. The biodistribution and limiting doses of AA139-nanomedicines were determined in uninfected rats via endotracheal aerosolization. The early bacterial killing activity of the AA139-nanomedicines in infected lungs was assessed in a rat model of pneumonia-septicemia caused by extended-spectrum β-lactamase-producing Klebsiella pneumoniae. In this model, the therapeutic efficacy was determined by once-daily (q24h) administration over 10 days. Both AA139-nanomedicines showed equivalent in vitro antimicrobial activities (similar to free AA139). In uninfected rats, they exhibited longer residence times in the lungs than free AA139 (∼20% longer for AA139-PNP and ∼80% longer for AA139-MCL), as well as reduced toxicity, enabling a higher limiting dose. In rats with pneumonia-septicemia, both AA139-nanomedicines showed significantly improved therapeutic efficacy in terms of an extended rat survival time, although survival of all rats was not achieved. These results demonstrate potential advantages that can be achieved using AMP-nanomedicines. AA139-PNP and AA139-MCL may be promising novel therapeutic agents for the treatment of patients suffering from multidrug-resistant Gram-negative pneumonia-septicemia.

scholarly journals Antibiotic resistance, biofilm formation, and biofilm-associated genes among Stenotrophomonas maltophilia clinical isolates

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Narjess Bostanghadiri ◽  
Abdollah Ardebili ◽  
Zohreh Ghalavand ◽  
Samane Teymouri ◽  
Mahsa Mirzarazi ◽  
...  
Keyword(s):  
Crystal Violet ◽  
Biofilm Formation ◽  
Staining Method ◽  
Clinical Isolates ◽  
Diffusion Method ◽  
Crystal Violet Staining ◽  
Disc Diffusion Method ◽  
Antimicrobial Susceptibility Pattern ◽  
Combination Strategies ◽  
Biofilm Production

Abstract Objective The purpose of the present study was to investigate the antimicrobial susceptibility pattern, biofilm production, and the presence of biofilm genes among the S. maltophilia clinical isolates. A total of 85 clinical isolates of S. maltophilia were collected from patients referred to several hospitals. Susceptibility to antibiotics was investigated by disc diffusion method according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI). By the crystal violet staining method, the capability of biofilm formation was examined. The genes associated with biofilm production were investigated by the PCR-sequencing techniques. Results All isolates were resistant to doripenem, imipenem, and meropenem. Minocycline, trimethoprim/sulfamethoxazole and levofloxacin exhibited the highest susceptibility of 100%, 97.65%, and 95.29%, respectively. The results of crystal violet staining assay showed that all isolates (100%) form biofilm. Moreover, 24 (28.23%), 32 (37.65%), and 29 (34.12%) of isolates were categorized as weak, moderate, and strong biofilm producers, respectively. Biofilm genes including rpfF, spgM and rmlA had an overall prevalence of 89.41% (76/85), 100% (85/85) and 84.71% (72/85), respectively. Rational prescribing of antibiotics and implementation of infection control protocols are necessary to prevent further infection and development of antimicrobial resistance. Combination strategies based on the appropriate antibiotics along with anti-biofilm agents can also be selected to eliminate biofilm-associated infections.

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