Therapeutic Potential of a Novel Lytic Phage, vB_EclM_ECLFM1, against Carbapenem-Resistant Enterobacter cloacae.

The global rise of multidrug-resistant Enterobacter cloacae strains, especially those that are resistant to carbapenems and produce metallo-ß-lactamases, poses a critical challenge in clinical settings owing to limited treatment options. While bacteriophages show promise in treating these infections, their use is hindered by scarce resources and insufficient genomic data. In this study, we isolated ECLFM1, a novel E. cloacae phage, from sewage water using a carbapenem-resistant clinical strain as the host. ECLFM1 exhibited rapid adsorption and a 15-min latent period, with a burst size of approximately 75 PFU/infected cell. Its genome, spanning 172,036 bp, was characterized and identified as a member of Karamvirus. In therapeutic applications, owing to a high multiplicity of infection, ECLFM1 showed increased survival in zebrafish infected with E. cloacae. This study highlights ECLFM1's potential as a candidate for controlling clinical E. cloacae infections, which would help address challenges in treating multidrug-resistant strains and contribute to the development of alternative treatments.

Isolation and characterization of bacteriophages with activities against multi-drug-resistant Acinetobacter nosocomialis causing bloodstream infection in vivo.

BACKGROUND: Acinetobacter nosocomialis (A. nosocomialis) is a glucose non-fermentative, gram-negative bacillus that belongs to the Acinetobacter calcoaceticus-baumannii complex. In recent years, studies have found an increased clinical prevalence of A. nosocomialis. However, given the increasing trend of antibiotic resistance, developing new antibacterial agents is vital. Currently, research regarding bacteriophage therapy against A. nosocomialis is only limited. METHODS: Two A. nosocomialis bacteriophages, TCUAN1 and TCUAN2, were isolated from sewage. Experiments such as transmission electron microscopy (TEM), host-range analysis, and sequencing were performed to determine their biological and genomic characteristics. TCUAN2 were further subjected to in vivo experiments and their derived-endolysin were cloned and tested against their bacteria host. RESULTS: Transmission electron microscopy revealed that TCUAN1 and TCUAN2 belong to Myoviridae and Podoviridae, respectively. Both phages show a broad host spectrum and rapid adsorption efficiency. Further biological analysis showed that TCUAN2 possesses a shorter latent period and larger burst size compared to TCUAN1. Because TCUAN2 showed a better antibacterial activity, it was injected into A. nosocomialis-infected mice which resulted in a significant decrease in bacterial load levels in the blood and increased the mice's survival. Finally, genomic analysis revealed that the complete nucleotide sequence of TCUAN1 is 49, 691 bps (containing 75 open reading frames) with a G + C content of 39.3%; whereas the complete nucleotide sequence of TCUAN2 is 41, 815 bps (containing 68 open reading frames) with a G + C content of 39.1%. The endolysin gene cloned and purified from TCUAN2 also showed antibacterial activity when used with a chelator EDTA.

Biological and genomic characterization of two newly isolated Elizabethkingia anophelis bacteriophages.

BACKGROUND: Elizabethkingia anophelis is an opportunistic pathogen that infects newborns and immunocompromised patients. Because the infection is associated with high mortality as a result of its intrinsic resistance to antibiotics, alternative treatment methods are needed. Our previous study successfully isolated the world's first E. anophelis phage, TCUEAP1, which showed beneficial protection to E. anophelis-infected mice. More new bacteriophages are needed in order to provide sufficient choices to combat E. anophelis infections. METHODS: In the current study, two new phages infecting E. anophelis were isolated from wastewater and were designated as TCUEAP2 and TCUEAP3. Further experiments, namely, transmission electron microscopy (TEM), infection assay, host-range analysis, and sequencing were performed to determine their biological and genomic characteristics. RESULTS: TEM analysis revealed that both TCUEAP2 and TCUEAP3 possess an icosahedral head with a non-contractile tail, and belong to the Siphoviridae family. Further experiments revealed that TCUEAP3 has a longer latent period and higher burst size compared to TCUEAP2. Host range analysis showed that both TCUEAP2 and TCUEAP3 have a narrow host range, infecting only their respective hosts. The genomic size of phage TCUEAP2 was 42,403 bps containing 61 predicted open reading frames (ORFs), whereas the genome size of TCUEAP3 was 37,073 bps containing 40 predicted ORFs. CONCLUSION: Due to the distinct biological characteristics of TCUEAP2 and TCUEAP3, they may be satisfactory for clinical uses such as preparation of phage cocktails or decontamination in clinical settings.

Rapid Identification of Methicillin-Resistant Staphylococcus aureus Using MALDI-TOF MS and Machine Learning from over 20,000 Clinical Isolates.

Rapidly identifying methicillin-resistant Staphylococcus aureus (MRSA) with high integration in the current workflow is critical in clinical practices. We proposed a matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS)-based machine learning model for rapid MRSA prediction. The model was evaluated on a prospective test and four external clinical sites. For the data set comprising 20,359 clinical isolates, the area under the receiver operating curve of the classification model was 0.78 to 0.88. These results were further interpreted using shapely additive explanations and presented using the pseudogel method. The important MRSA feature, m/z 6,590 to 6,599, was identified as a UPF0337 protein SACOL1680 with a lower binding affinity or no docking results compared with UPF0337 protein SA1452, which is mainly detected in methicillin-susceptible S. aureus. Our MALDI-TOF MS-based machine learning model for rapid MRSA identification can be easily integrated into the current clinical workflows and can further support physicians in prescribing proper antibiotic treatments. IMPORTANCE Over 20,000 clinical MSSA and MRSA isolates were collected to build a machine learning (ML) model to identify MSSA/MRSA and their markers. This model was tested across four external clinical sites to ensure the model's usability. We report the first discovery and validation of MRSA markers on the largest scale of clinical MSSA and MRSA isolates collected to date, covering five different clinical sites. Our developed approach for the rapid identification of MSSA and MRSA can be highly integrated into the current workflows.

Respiratory pathogens - Some altered antibiotic susceptibility after implementation of pneumococcus vaccine and antibiotic control strategies.

BACKGROUND/PURPOSE: Antimicrobial resistance in Taiwan has been on the rise for two decades. The implementation of pneumococcal conjugate vaccination (PCV13) and enhanced antimicrobial control (2013-2015) by the government may have changed the antibiotic resistance. METHODS: Four respiratory pathogens (Streptococcus pneumoniae, Haemophilus influenzae, Streptococcus pyogenes, and Moraxella catarrhalis) isolated in a single medical center during 2008-2017 were studied. We defined three temporal stages: (a) the first era (2008-2012), prior to implementation of the national immunization program (PCV13 vaccination), (b) the second era (2013-2015), during which an enhanced antibiotic control strategy was implemented, and (c) the third era (2016-2017), after implementation. Antimicrobial drug sensitivities were collected from two other hospitals: one from east Taiwan, one from west-central Taiwan. RESULTS: S. pneumoniae was frequently isolated during the first era. It declined progressively during the second era of PCV13 vaccination. S. pyogenes and M. catarrhalis were not frequently isolated. The drug susceptibility of S. pneumoniae to ceftriaxone and vancomycin remained high. The antimicrobial susceptibility of H. influenzae to amoxillin/clavulante declined over the three temporal stages, from 91.9%-79.5%-58.5% (all p < 0.05). CONCLUSION: Antimicrobial resistance of H. influenzae increased during the latter part of the study period. The PCV13 vaccination program reduced the invasive pneumococcal disease and reduced the stress on the emergent drug resistance. This enhanced antibiotic control strategy was effective in terms of nosocomial drug resistance but not for community-associated pathogens.

Phosphoenolpyruvate phosphotransferase system components positively regulate Klebsiella biofilm formation.

BACKGROUND/PURPOSE: Klebsiella pneumoniae is one of the leading causes of device-related infections (DRIs), which are associated with attachment of bacteria to these devices to form a biofilm. The latter is composed of not only bacteria but also extracellular polymeric substances (EPSes) consisting of extracellular DNAs, polysaccharides, and other macromolecules. The phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS) regulates diverse processes of bacterial physiology. In the genome of K. pneumoniae MGH 78578, we found an uncharacterized enzyme II complex homolog of PTS: KPN00353 (EIIA homolog), KPN00352 (EIIB homolog), and KPN00351 (EIIC homolog). The aim of this study was to characterize the potential physiological role of KPN00353, KPN00352, and KPN00351 in biofilm formation by K. pneumoniae. METHODS/RESULTS: We constructed the PTS mutants and recombinant strains carrying the gene(s) of PTS. The recombinant K. pneumoniae strain overexpressing KPN00353-KPN00352-KPN00351 produced more extracellular matrix than did the vector control according to transmission and scanning electron microscopy. Judging by quantification of biofilm formation, of extracellular DNA (eDNA), and of capsular polysaccharide, the recombinant strain overexpressing KPN00353-KPN00352-KPN00351 produced more biofilm and capsular polysaccharide after overnight culture and more eDNA in the log phase as compared to the vector control. CONCLUSION: The genes, KPN00353-KPN00352-KPN00351, encode a putative enzyme II complex in PTS and positively regulate biofilm formation by enhancing production of eDNA and capsular polysaccharide in K. pneumoniae. Five proteins related to chaperones, to the citric acid cycle, and to quorum sensing are upregulated by the KPN00353-KPN00352-KPN00351 system.

Use of a Sampling Area-Adjusted Adenosine Triphosphate Bioluminescence Assay Based on Digital Image Quantification to Assess the Cleanliness of Hospital Surfaces.

Contaminated surfaces play an important role in the transmission of pathogens. We sought to establish a criterion that could indicate "cleanliness" using a sampling area-adjusted adenosine triphosphate (ATP) assay. In the first phase of the study, target surfaces were selected for swab sampling before and after daily cleaning; then, an aerobic colony count (ACC) plate assay of bacteria and antibiotic-resistant bacteria was conducted. ATP swabs were also tested, and the ATP readings were reported as relative light units (RLUs). The results of the ACC and ATP assays were adjusted according to the sampling area. During the second phase of the study, a new cleaning process employing sodium dichloroisocyanurate (NaDCC) was implemented for comparison. Using the criterion of 2.5 colony-forming units (CFU)/cm², 45% of the sampled sites were successfully cleaned during phase one of the study. During phase two, the pass rates of the surface samples (64%) were significantly improved, except under stringent (5 RLU/cm²) and lax (500 RLU) ATP criteria. Using receiver-operating characteristic curve analysis, the best cut-off point for an area-adjusted ATP level was 7.34 RLU/cm², which corresponded to culture-assay levels of <2.5 CFU/cm². An area adjustment of the ATP assay improved the degree of correlation with the ACC-assay results from weak to moderate.

Rapid and sensitive detection of Acinetobacter baumannii using loop-mediated isothermal amplification.

Here we report the design and evaluation of a loop-mediated isothermal amplification (LAMP) assay for detecting Acinetobacter baumannii DNA based on the 16S-23S rRNA intergenic spacer (ITS) sequence. The results showed that target DNA was amplified and visualized within 30min and with a detection limit 100-fold greater than PCR.