Molecular Detection of Carbapenemases in Acinetobacter baumannii Strains of Portugal and Association With Sequence Types, Capsular Types, and Virulence.

PURPOSE: Carbapenem-resistant Acinetobacter baumannii (CRAB) is an important nosocomial pathogen. The capsular type (K-type) is considered a major virulence factor, contributing to the evasion of host defenses. The global spread and dissemination dynamics between K-types, sequence types (ST), antibiotic resistance genes, and virulence factors remain largely unknown in Portugal. METHODS: A collection of 96 CRAB clinical samples collected between 2005 and 2019 in the northern region of Portugal were tested for antimicrobial susceptibility profile and screened by polymerase chain reaction for resistance genetic determinants. A subset of 26 representative isolates was subjected to whole-genome sequencing to assess K types, ST types, and genomic relatedness. The pathogenicity of distinct K-types was also tested using Galleria mellonella model. FINDINGS: For the 96 CRAB isolates analyzed, high antimicrobial resistance (>90%) was observed to the carbapenems, fluoroquinolones, and miscellaneous agents. Greater antimicrobial susceptibility (∼30%-57%) was observed for aminoglycosides, particularly tobramycin, and amikacin. Genotypically, 75 strains (78.5%) carried blaOXA-23-like, 18 strains (18.8%) carried blaIMP-like, and 11 strains (14.9%) carried blaOXA-40-like carbapenem resistance genes, respectively. Associations between OXA and ST/capsular locus (KL) types were observed over the years (eg, OXA-40-like/ST46Past/KL120 and OXA-23-like/ST2Past/KL2). ST2Past of clonal complex II was present in most strains, a dominant drug-resistant lineage in the United States and Europe. KL7 was also the most prevalent KL-type (38.5%), followed by KL2 (34.6%), KL120 (23.1%), and KL9 (3.8%). Virulence assessment for different K-types in a Galleria mellonella model revealed a significantly increased virulence for KL120 when compared with KL7, KL9, and KL2. IMPLICATIONS: There are specific CRAB serotypes circulating in Portugal, accounting by the low diversity of acquired carbapenemase genes (OXA-23-like and OXA-40-like), ST types (ST2 and ST46) and KL types (KL2, KL7, KL9, and KL120) identified. The high prevalent of ST2, especially when associated with KL2 and blaOXA-23-like, suggest that antibiotic resistance has been driven by clonal expansion of clonal complex II. Such findings provide useful information on the diversity of multidrug-resistant bacterium that might be relevant for antibacterial interventions.

In Vitro and In Vivo Assessments of Newly Isolated N4-like Bacteriophage against ST45 K62 Capsular-Type Carbapenem-Resistant Klebsiella pneumoniae: vB_kpnP_KPYAP-1.

The rise of carbapenem-resistant Klebsiella pneumoniae (CRKP) presents a significant global challenge in clinical and healthcare settings, severely limiting treatment options. This study aimed to utilize a bacteriophage as an alternative therapy against carbapenem-resistant K. pneumoniae. A novel lytic N4-like Klebsiella phage, vB_kpnP_KPYAP-1 (KPYAP-1), was isolated from sewage. It demonstrated efficacy against the K62 serotype polysaccharide capsule of blaOXA-48-producing K. pneumoniae. KPYAP-1 forms small, clear plaques, has a latent period of 20 min, and reaches a growth plateau at 35 min, with a burst size of 473 plaque-forming units (PFUs) per infected cell. Phylogenetic analysis places KPYAP-1 in the Schitoviridae family, Enquatrovirinae subfamily, and Kaypoctavirus genus. KPYAP-1 employs an N4-like direct terminal repeat mechanism for genome packaging and encodes a large virion-encapsulated RNA polymerase. It lacks integrase or repressor genes, antibiotic resistance genes, bacterial virulence factors, and toxins, ensuring its safety for therapeutic use. Comparative genome analysis revealed that the KPYAP-1 genome is most similar to the KP8 genome, yet differs in tail fiber protein, indicating variations in host recognition. In a zebrafish infection model, KPYAP-1 significantly improved the survival rate of infected fish by 92% at a multiplicity of infection (MOI) of 10, demonstrating its potential for in vivo treatment. These results highlight KPYAP-1 as a promising candidate for developing phage-based therapies targeting carbapenemase-producing K. pneumoniae.

Molecular and phenotypic characterization of Streptococcus pneumoniae isolates in a Japanese tertiary care hospital.

This study aimed to investigate the bacterial characteristics of pneumococcal isolates obtained from a tertiary care hospital in Japan. We analyzed the antimicrobial susceptibility, possession of macrolide resistance genes, pneumococcal serogroup/serotype, and sequence type (ST) of pneumococcal isolates from patients aged 15 years or older between 2011 and 2020 at Nagasaki University Hospital. Of the 73 isolates analyzed, 86.3% showed resistance to macrolides, and 28.8%, 46.6%, and 11.0% harbored mefA, ermB, and both, respectively. Of the isolates possessing ermB, 97.6% showed high levels of macrolide resistance [minimal inhibitory concentration (MIC) range, > 16 µg/mL]. Solithromycin (MIC range, 0.03-0.25 µg/mL), regardless of the presence of macrolide resistance genes, and lascufloxacin (MIC range, 0.06-0.5 µg/mL) showed potent in vitro activity against pneumococci. Serotype 19A was the most prevalent (six isolates), followed by serotypes 10A, 15A, and 15B/C (five isolates each). Four serotypes (11A, 19A, 22F, and 23B) and five STs (36, 99, 433, 558, and 3111) were significantly correlated with the presence of macrolide resistance genes. All four isolates with serotype 11A/ST99 and three isolates with serotype 19A/ST3111 harbored both mefA and ermB. No macrolide resistance genes were detected in either of the two isolates with serotype 22F/ST433, while all ten isolates with serogroup 15 (serotypes 15A and 15B/C, five isolates each) possessed ermB alone. Our study revealed the bacterial characteristics of the pneumococcal isolates obtained from our hospital. In vitro activity of solithromycin and lascufloxacin against these isolates was confirmed.

Lytic Capsule-Specific Acinetobacter Bacteriophages Encoding Polysaccharide-Degrading Enzymes.

The genus Acinetobacter comprises both environmental and clinically relevant species associated with hospital-acquired infections. Among them, Acinetobacter baumannii is a critical priority bacterial pathogen, for which the research and development of new strategies for antimicrobial treatment are urgently needed. Acinetobacter spp. produce a variety of structurally diverse capsular polysaccharides (CPSs), which surround the bacterial cells with a thick protective layer. These surface structures are primary receptors for capsule-specific bacteriophages, that is, phages carrying tailspikes with CPS-depolymerizing/modifying activities. Phage tailspike proteins (TSPs) exhibit hydrolase, lyase, or esterase activities toward the corresponding CPSs of a certain structure. In this study, the data on all lytic capsule-specific phages infecting Acinetobacter spp. with genomes deposited in the NCBI GenBank database by January 2024 were summarized. Among the 149 identified TSPs encoded in the genomes of 143 phages, the capsular specificity (K specificity) of 46 proteins has been experimentally determined or predicted previously. The specificity of 63 TSPs toward CPSs, produced by various Acinetobacter K types, was predicted in this study using a bioinformatic analysis. A comprehensive phylogenetic analysis confirmed the prediction and revealed the possibility of the genetic exchange of gene regions corresponding to the CPS-recognizing/degrading parts of different TSPs between morphologically and taxonomically distant groups of capsule-specific Acinetobacter phages.

Identification and characterization of the capsule depolymerase Dpo27 from phage IME-Ap7 specific to Acinetobacter pittii.

Among the Acinetobacter genus, Acinetobacter pittii stands out as an important opportunistic infection causative agent commonly found in hospital settings, which poses a serious threat to human health. Recently, the high prevalence of carbapenem-resistant A. pittii isolates has created significant therapeutic challenges for clinicians. Bacteriophages and their derived enzymes are promising therapeutic alternatives or adjuncts to antibiotics effective against multidrug-resistant bacterial infections. However, studies investigating the depolymerases specific to A. pittii strains are scarce. In this study, we identified and characterized a capsule depolymerase, Dpo27, encoded by the bacteriophage IME-Ap7, which targets A. pittii. A total of 23 clinical isolates of Acinetobacter spp. were identified as A. pittii (21.91%, 23/105), and seven A. pittii strains with various K locus (KL) types (KL14, KL32, KL38, KL111, KL163, KL207, and KL220) were used as host bacteria for phage screening. The lytic phage IME-Ap7 was isolated using A. pittii 7 (KL220) as an indicator bacterium and was observed for depolymerase activity. A putative tail fiber gene encoding a polysaccharide-degrading enzyme (Dpo27) was identified and expressed. The results of the modified single-spot assay showed that both A. pittii 7 and 1492 were sensitive to Dpo27, which was assigned the KL220 type. After incubation with Dpo27, A. pittii strain was susceptible to killing by human serum; moreover, the protein displayed no hemolytic activity against erythrocytes. Furthermore, the protein exhibited sustained activity across a wide pH range (5.0-10.0) and at temperatures between 20 and 50°C. In summary, the identified capsule depolymerase Dpo27 holds promise as an alternative treatment for combating KL220-type A. pittii infections.

Molecular characterization of Pasteurella multocida from cats and antibiotic sensitivity of the isolates.

BACKGROUND: Companion animals, including dogs and cats, are frequently identified as sources of Pasteurella multocida, a bacterium that can be transmitted to humans and cause infections. OBJECTIVES: This survey defines the prevalence, antibiotic sensitivity, capsular types, lipopolysaccharide (LPS) types and virulence factors of P. multocida isolated from cats. METHODS: A total of 100 specimens from various cat breeds were collected. P. multocida was characterized using both biochemical tests and PCR. Genotypes of isolates were determined using capsular and LPS typing methods. Additionally, virulotyping was performed by detecting the presence of 12 virulence-associated genes. Disk diffusion was used to determine the antibiotic sensitivity of the isolates. RESULTS: The prevalence of P. multocida in cats was 29%. Among the isolates, the majority were capsular type A (96.5%) and type D (3.4%), with a predominant presence of type A. Twenty-six of the isolates (89.66%) belonged to LPS genotype L6, whereas three isolates (10.3%) belonged to genotype L3. Among the 12 virulence genes examined, sodC, oma87, ptfA, nanB and ompH showed remarkable prevalence (100%). The toxA gene was detected in four isolates (13.8%). Variations were observed in other virulence genes. The nanH gene was present in 93.1% of the isolates, whereas the pfhA gene was detected in 58.6% of the isolates. The exbD-tonB, hgbB, sodA and hgbA genes showed prevalence rates of 96.5%, 96.5%, 96.5% and 82.8%, respectively. Additionally, particular capsule and LPS types were associated with specific virulence genes. Specifically, the toxA and pfhA genes were found to be more prevalent in isolates with capsular type A and LPS genotype L6. Most isolates were resistant to ampicillin, clindamycin, lincomycin, streptomycin and penicillin. CONCLUSIONS: According to this epidemiological and molecular data, P. multocida from cats possess several virulence-associated genes and are resistant to antimicrobial medicines commonly used in humans and animals. Thus, it is crucial to consider the public health concerns of P. multocida in humans.

Molecular evaluation of sheep and goats isolates of Pasteurella multocida and their antibiotic resistance.

Pasteurella multocida exists as a commensal in the upper respiratory tracts of livestock, and poultry, and causes a wide variety of diseases in humans and animals. This study aimed to investigate the incidence of P. multocida by bacteriological and molecular characterization in sheep and goats and screening the existence of capsule-specific genes and their antibiotic resistance pattern. Totally, 1650 nasopharyngeal swabs were collected from apparently healthy sheep and goats and 460 lung tissues were collected from slaughtered animals in Fars province, Iran. All samples were cultured and suspected colonies were examined by biochemical tests, antimicrobial assay and polymerase chain reaction (PCR). Among 165 P. multocida (104 sheep and 61 goats) isolates, the capA, capD, and capB genes were amplified in 98, 48, and 12 isolates, respectively. The occurrence of four virulence-associated genes of P. multocida isolates were determined by PCR. Most isolates harbored the toxA (79.40%) and hgbB genes (70.90%) and 59.40% of isolates had the pfhA gene. Almost half of the isolates (46.10%) contained the tbpA gene. According to the current study, P. multocida capsular type A had the most frequency followed by type D. In addition, the high frequency of tbpA, pfhA, toxA, and hgbB genes revealed that these genes are possibly important in the pathogenesis of P. multocida. Oxytetracycline, enrofloxacin, florfenicol, and tilmicosin were the most effective drugs.

Structural biology and functional features of phage-derived depolymerase Depo32 on Klebsiella pneumoniae with K2 serotype capsular polysaccharides.

Hypervirulent Klebsiella pneumoniae with capsular polysaccharides (CPSs) causes severe nosocomial- and community-acquired infections. Phage-derived depolymerases can degrade CPSs from K. pneumoniae to attenuate bacterial virulence, but their antimicrobial mechanisms and clinical potential are not well understood. In the present study, Klebsiella phage GH-K3-derived depolymerase Depo32 (encoded by gene gp32) was identified to exhibit high efficiency in specifically degrading the CPSs of K2 serotype K. pneumoniae. The cryo-electron microscopy structure of trimeric Depo32 at a resolution up to 2.32 Å revealed potential catalytic centers in the cleft of each of the two adjacent subunits. K. pneumoniae subjected to Depo32 became more sensitive to phagocytosis by RAW264.7 cells and activated the cells by the mitogen-activated protein kinase signaling pathway. In addition, intranasal inoculation with Depo32 (a single dose of 200 µg, 20 µg daily for 3 days, or in combination with gentamicin) rescued all C57BL/6J mice infected with a lethal dose of K. pneumoniae K7 without interference from its neutralizing antibody. In summary, this work elaborates on the mechanism by which Depo32 targets the degradation of K2 serotype CPSs and its potential as an antivirulence agent. IMPORTANCE Depolymerases specific to more than 20 serotypes of Klebsiella spp. have been identified, but most studies only evaluated the single-dose treatment of depolymerases with relatively simple clinical evaluation indices and did not reveal the anti-infection mechanism of these depolymerases in depth. On the basis of determining the biological characteristics, the structure of Depo32 was analyzed by cryo-electron microscopy, and the potential active center was further identified. In addition, the effects of Depo32 on macrophage phagocytosis, signaling pathway activation, and serum killing were revealed, and the efficacy of the depolymerase (single treatment, multiple treatments, or in combination with gentamicin) against acute pneumonia caused by Klebsiella pneumoniae was evaluated. Moreover, the roles of the active sites of Depo32 were also elucidated in the in vitro and in vivo studies. Therefore, through structural biology, cell biology, and in vivo experiments, this study demonstrated the mechanism by which Depo32 targets K2 serotype K. pneumoniae infection.

Friunavirus Phage-Encoded Depolymerases Specific to Different Capsular Types of Acinetobacter baumannii.

Acinetobacter baumannii is a critical priority nosocomial pathogen that produces a variety of capsular polysaccharides (CPSs), the primary receptors for specific depolymerase-carrying phages. In this study, the tailspike depolymerases (TSDs) encoded in genomes of six novel Friunaviruses, APK09, APK14, APK16, APK86, APK127v, APK128, and one previously described Friunavirus phage, APK37.1, were characterized. For all TSDs, the mechanism of specific cleavage of corresponding A. baumannii capsular polysaccharides (CPSs) was established. The structures of oligosaccharide fragments derived from K9, K14, K16, K37/K3-v1, K86, K127, and K128 CPSs degradation by the recombinant depolymerases have been determined. The crystal structures of three of the studied TSDs were obtained. A significant reduction in mortality of Galleria mellonella larvae infected with A. baumannii of K9 capsular type was shown in the example of recombinant TSD APK09_gp48. The data obtained will provide a better understanding of the interaction of phage-bacterial host systems and will contribute to the formation of principles of rational usage of lytic phages and phage-derived enzymes as antibacterial agents.

First study on capsular serotypes and virulence factors of Pasteurella multocida isolates from Phan Rang sheep in Vietnam.

Background and Aim: Pasteurella multocida is considered as a main factor mediating pneumonic pasteurellosis in ruminants, including sheep. It is also a current threat to Phan Rang sheep in Vietnam. This study aimed to characterize P. multocida isolated from Phan Rang sheep, their antibiotic resistance profile, and the prevalence of some virulence-associated genes of these strains. Materials and Methods: Bacteria were isolated on brain heart infusion, 10% sheep blood agar plates, and screened by biochemical tests. The polymerase chain reaction technique was used with specific primers to identify P. multocida, the presence of virulence-associated genes, and serotypes of isolates. Antimicrobial susceptibility and biofilm formation of isolates were examined using the disk diffusion method and crystal violet-based method, respectively. Results: A total of 41 P. multocida strains were isolated from 485 samples from clinically sick and healthy sheep. Of the isolates, 58.53% were serotype A, 9.75% were serotype B, and 31.71% were serotype D. Healthy animals were infected with serotype D only. All 15 virulence genes were identified in all strains isolated from clinically sick sheep, while strains isolated from healthy sheep carried 11/15 virulence genes tested. Among virulence-associated genes exbB, exbD, tonB, ompA, oma87, fimA, hgbA, and nanB were detected in over 90% of isolates, whereas hgbB, nanH, tbpA and pfhA were less frequent. Interestingly, pmHAS and tadD were highly prevalent in capsular type A strains, whereas the toxA gene was detected in capsular type D strains only. All of the isolated strains were fully susceptible to enrofloxacin, ciprofloxacin, neomycin, and ofloxacin. About 92.68% were susceptible to chloramphenicol and 90.24% to amikacin, but there was high resistance to erythromycin, tetracycline, and amoxicillin. Our results reveal that 53.65% of 41 isolates could produce biofilm, whereas 46.34% could not. Conclusion: Pasteurella multocida from Phan Rang sheep possess many virulence genes and resistance to several common antibiotics such as erythromycin, tetracycline, and amoxicillin. The results are an important warning regarding antibiotic resistance of P. multocida.

Relationship between Biofilm Production and High Somatic Cell Count in Streptococcus agalactiae Isolated from Milk of Cows with Subclinical Mastitis.

Streptococcus agalactiae (S. agalactiae) is one of the main agents that causes mastitis in dairy cows, mainly inducing the subclinical form, which is characterized by a high somatic cell count (SCC). The aim of this study was to correlate the increase in SCC caused by S. agalactiae in cows with subclinical mastitis to the presence of genes related to adhesion and invasion in bovine mammary epithelial cells (BMEC) and biofilm formation. Considering the 145 isolates tested, 57.2% presented the capsular type Ia and 42.8% presented type III. We identified the virulence genes among the isolates and determined nine genetic profiles. The most common profile was identified in 69 isolates (47.5%): Ia, fbsA+, fbsB-, pI1-, pI2a-, pI2b+, and hylb+. All isolates produced biofilm, with 58.6% classified as strong producers, 29% as moderate producers and 12.4% as weak producers. No statistical correlation was found between the presence of virulence genes and increased SCC or biofilm production. However, biological evidence was observed between increased SCC and biofilm production. One isolate from each profile was randomly subjected to adhesion and invasion assays, and all of them adhered to BEMC, but none were able to invade. Our results showed that different genetic profiles do not provide advantages for bacteria to invade BMEC in vitro. In addition, biofilm production appears to be related to high SCC.

Isolation and Characterization of the First Zobellviridae Family Bacteriophage Infecting Klebsiella pneumoniae.

In order to address the upcoming crisis in the treatment of Klebsiella pneumoniae infections, caused by an increasing proportion of resistant isolates, new approaches to antimicrobial therapy must be developed. One approach would be to use (bacterio)phages and/or phage derivatives for therapy. In this study, we present a description of the first K. pneumoniae phage from the Zobellviridae family. The vB_KpnP_Klyazma podovirus, which forms translucent halos around the plaques, was isolated from river water. The phage genome is composed of 82 open reading frames, which are divided into two clusters located on opposite strands. Phylogenetic analysis revealed that the phage belongs to the Zobellviridae family, although its identity with the closest member of this family was not higher than 5%. The bacteriophage demonstrated lytic activity against all (n = 11) K. pneumoniae strains with the KL20 capsule type, but only the host strain was lysed effectively. The receptor-binding protein of the phage was identified as a polysaccharide depolymerase with a pectate lyase domain. The recombinant depolymerase protein showed concentration-dependent activity against all strains with the KL20 capsule type. The ability of a recombinant depolymerase to cleave bacterial capsular polysaccharides regardless of a phage's ability to successfully infect a particular strain holds promise for the possibility of using depolymerases in antimicrobial therapy, even though they only make bacteria sensitive to environmental factors, rather than killing them directly.

Invasive Haemophilus influenzae disease among adults in Japan during 2014-2018.

PURPOSE: We describe the epidemiology of invasive Haemophilus influenzae disease (IHD) among adults in Japan. METHODS: Data for 200 adult IHD patients in 2014-2018 were analyzed. The capsular type of H. influenzae was determined by bacterial agglutination and polymerase chain reaction (PCR), and non-typeable Haemophilus influenzae (NTHi) was identified by PCR. RESULTS: The annual incidence of IHD (cases per 100,000 population) was 0.12 for age 15-64 years and 0.88 for age ≥ 65 years in 2018. The median age was 77 years, and 73.5% were aged ≥ 65 years. About one-fourth of patients were associated with immunocompromising condition. The major presentations were pneumonia, followed by bacteremia, meningitis and other than pneumonia or meningitis (other diseases). The case fatality rate (CFR) was 21.2% for all cases, and was significantly higher in the ≥ 65-year group (26.1%) than in the 15-64-year group (7.5%) (p = 0.013). The percentage of cases with pneumonia was significantly higher in the ≥ 65-year group than in the 15-64-year group (p < 0.001). The percentage of cases with bacteremia was significantly higher in the 15-64-year group than in the ≥ 65-year group (p = 0.027). Of 200 isolates, 190 (95.0%) were NTHi strains, and the other strains were encapsulated strains. 71 (35.5%) were resistant to ampicillin, but all were susceptible to ceftriaxone. CONCLUSION: The clinical presentations of adult IHD patients varied widely; about three-fourths of patients were age ≥ 65 years and their CFR was high. Our findings support preventing strategies for IHD among older adults, including the development of NTHi vaccine.

Identification of a phage-derived depolymerase specific for KL47 capsule of Klebsiella pneumoniae and its therapeutic potential in mice.

Klebsiella pneumoniae is one of the major pathogens causing global multidrug-resistant infections. Therefore, strategies for preventing and controlling the infections are urgently needed. Phage depolymerase, often found in the tail fiber protein or the tail spike protein, is reported to have antibiofilm activity. In this study, phage P560 isolated from sewage showed specific for capsule locus type KL47 K. pneumoniae, and the enlarged haloes around plaques indicated that P560 encoded a depolymerase. The capsule depolymerase, ORF43, named P560dep, derived from phage P560 was expressed, purified, characterized and evaluated for enzymatic activity as well as specificity. We reported that the capsule depolymerase P560dep, can digest the capsule polysaccharides on the surface of KL47 type K. pneumoniae, and the depolymerization spectrum of P560dep matched to the host range of phage P560, KL47 K. pneumoniae. Crystal violet staining assay showed that P560dep was able to significantly inhibit biofilm formation. Further, a single dose (50 µg/mouse) of depolymerase intraperitoneal injection protected 90%-100% of mice from lethal challenge before or after infection by KL47 carbapenem-resistant K. pneumoniae. And pathological changes were alleviated in lung and liver of mice infected by KL47 type K. pneumoniae. It is demonstrated that depolymerase P560dep as an attractive antivirulence agent represents a promising tool for antimicrobial therapy.

Capsule-Targeting Depolymerases Derived from Acinetobacter baumannii Prophage Regions.

In this study, several different depolymerases encoded in the prophage regions of Acinetobacter baumannii genomes have been bioinformatically predicted and recombinantly produced. The identified depolymerases possessed multi-domain structures and were identical or closely homologous to various proteins encoded in other A. baumannii genomes. This means that prophage-derived depolymerases are widespread, and different bacterial genomes can be the source of proteins with polysaccharide-degrading activities. For two depolymerases, the specificity to capsular polysaccharides (CPSs) of A. baumannii belonging to K1 and K92 capsular types (K types) was determined. The data obtained showed that the prophage-derived depolymerases were glycosidases that cleaved the A. baumannii CPSs by the hydrolytic mechanism to yield monomers and oligomers of the K units. The recombinant proteins with established enzymatic activity significantly reduced the mortality of Galleria mellonella larvae infected with A. baumannii of K1 and K92 capsular types. Therefore, these enzymes can be considered as suitable candidates for the development of new antibacterials against corresponding A. baumannii K types.

Isolation and Characterisation of Bacteriophage Selective for Key Acinetobacter baumannii Capsule Chemotypes.

Nineteen bacteriophages against five main capsular types of multidrug-resistant Acinetobacter baumannii were isolated from tertiary care hospital sewage. Eight representative phages from each capsular type were characterized and tested for their biological properties. The biological features revealed that phages T1245, T444, and T515 had a large burst size of more than 420 pfu/mL, together with a short latent period lasting less than 6 min, and were readily adsorbed to a bacterial host within 10 min. Moreover, these phages demonstrated host specificity and stability over a broad range of temperatures (-20 to 60 °C) and pH (5.0-9.0). A whole-genome analysis of six lytic and two temperate phages revealed high genomic similarity with double-stranded DNA between 40 and 50 kb and G + C content of 38-39%. The protein compositions disclosed the absence of toxin-coding genes. The phylogenic results, together with morphological micrographs, confirmed that three selected phages (T1245, T444, and T515) belong to the Podoviridae family within the order Caudovirales. The biological data and bioinformatics analysis indicated that these novel A. baumannii phages possess important enzymes, including depolymerase and endolysin, which could be further developed as promising alternative antibacterial agents to control A. baumannii infections.

Epidemiological features of nosocomial Klebsiella pneumoniae: virulence and resistance determinants.

Aim: The authors aimed to examine antibiotic resistance genes and representative virulence determinants among 100 Klebsiella pneumoniae isolates with an emphasis on capsular serotypes and clonality of some of the isolates. Methods: PCR amplification of (rmpA, rmpA2, iutA, iroN and IncHI1B plasmid) and (NDM, OXA-48, KPC, CTX-M-15, VIM, IMP, SPM) was conducted. Wzi sequencing and multilocus sequence typing (MLST) were performed. Results: K2 was the only detected serotype in the authors' collection. RMPA2 was the most common capsule-associated virulence gene detected. All studied isolates harbored OXA-48-like (100%) and NDM (43%) (n = 43). ST147 was the most common sequence type. Conclusion: This work provides insight into the evolution of the coexistence of virulence and resistance genes in a tertiary healthcare setting in Cairo, Egypt.

Exploring the association between capsular types, sequence types, and carbapenemase genes in Acinetobacter baumannii.

Acinetobacter baumannii is the main cause of nosocomial infections, which are increasingly difficult to treat due to the emergence of carbapenem resistance. This study focused on major carbapenemase genes and explored the association between carbapenemase genes, sequence types (ST types), and capsular types (K types). A total of 98 carbapenem-resistant A. baumannii (CRAB) strains were collected from two hospitals, the Chang Gung Memorial Hospital-Lin Kou branch (LCGMH) in northern Taiwan and the CGMH-Kaohsiung branch (KCGMH) in southern Taiwan, from 2015 to 2017. Major carbapenemase genes of class A, B, and D ß-lactamases were detected by polymerase chain reaction. All strains except 1 were positive for blaOXA-51-like, 76 strains (77.6%) carried blaOXA-23-like, and 25 strains (25.5%) carried blaOXA-24-like. The regional distribution showed that blaOXA-23-like was more common than blaOXA-24-like in both hospitals (85.3% and 60% in LCGMH and KCGMH, respectively); however, the percentage of blaOXA-24-like was much higher in KCGMH (46.7%) than in LCGMH (16.2%). Oxford multilocus sequence typing and global optimal eBURST analysis were conducted for 59 strains. The results of this study showed the association between blaOXA gene patterns, ST types, and K types and demonstrated that four major K types, KL2, KL10, KL22, and KL52, which were associated with specific ST types, were mainly clustered into clonal complexes CC208 and CC549 (a unique clonal complex found in Taiwan). These findings provide important information for monitoring the epidemiology and dissemination of this pathogen.

Novel Acinetobacter baumannii Bacteriophage Aristophanes Encoding Structural Polysaccharide Deacetylase.

Acinetobacter baumannii appears to be one of the most crucial nosocomial pathogens. A possible component of antimicrobial therapy for infections caused by extremely drug-resistant A. baumannii strains may be specific lytic bacteriophages or phage-derived enzymes. In the present study, we observe the biological features, genomic organization, and phage-host interaction strategy of novel virulent bacteriophage Aristophanes isolated on A. baumannii strain having K26 capsular polysaccharide structure. According to phylogenetic analysis phage Aristophanes can be classified as a representative of a new distinct genus of the subfamily Beijerinckvirinae of the family Autographiviridae. This is the first reported A. baumannii phage carrying tailspike deacetylase, which caused O-acetylation of one of the K26 sugar residues.

Multidrug-Resistant Klebsiella pneumoniae Causing Severe Infections in the Neuro-ICU.

The purpose of this study was the identification of genetic lineages and antimicrobial resistance (AMR) and virulence genes in Klebsiella pneumoniae isolates associated with severe infections in the neuro-ICU. Susceptibility to antimicrobials was determined using the Vitek-2 instrument. AMR and virulence genes, sequence types (STs), and capsular types were identified by PCR. Whole-genome sequencing was conducted on the Illumina MiSeq platform. It was shown that K. pneumoniae isolates of ST14K2, ST23K57, ST39K23, ST76K23, ST86K2, ST218K57, ST219KL125/114, ST268K20, and ST2674K47 caused severe systemic infections, including ST14K2, ST39K23, and ST268K20 that were associated with fatal incomes. Moreover, eight isolates of ST395K2 and ST307KL102/149/155 were associated with manifestations of vasculitis and microcirculation disorders. Another 12 K. pneumoniae isolates of ST395K2,KL39, ST307KL102/149/155, and ST147K14/64 were collected from patients without severe systemic infections. Major isolates (n = 38) were XDR and MDR. Beta-lactamase genes were identified: blaSHV (n = 41), blaCTX-M (n = 28), blaTEM (n = 21), blaOXA-48 (n = 21), blaNDM (n = 1), and blaKPC (n = 1). The prevalent virulence genes were wabG (n = 41), fimH (n = 41), allS (n = 41), and uge (n = 34), and rarer, detected only in the genomes of the isolates causing severe systemic infections-rmpA (n = 8), kfu (n = 6), iroN (n = 5), and iroD (n = 5) indicating high potential of the isolates for hypervirulence.