Phage-specific antibodies: are they a hurdle for the success of phage therapy?

Phage therapy has attracted attention again owing to the increasing number of drug-resistant bacteria. Although the efficacy of phage therapy has been reported, numerous studies have indicated that the generation of phage-specific antibodies resulting from phage administration might have an impact on clinical outcomes. Phage-specific antibodies promote phage uptake by macrophages and contribute to their rapid clearance from the body. In addition, phage-specific neutralizing antibodies bind to the phages and diminish their antibacterial activity. Thus, phage-specific antibody production and its role in phage therapy have been analyzed both in vitro and in vivo. Strategies for prolonging the blood circulation time of phages have also been investigated. However, despite these efforts, the results of clinical trials are still inconsistent, and a consensus on whether phage-specific antibodies influence clinical outcomes has not yet been reached. In this review, we summarize the phage-specific antibody production during phage therapy. In addition, we introduce recently performed clinical trials and discuss whether phage-specific antibodies affect clinical outcomes and what we can do to further improve phage therapy regimens.

Identification of a mycobacterial hydrazidase, an isoniazid-hydrolyzing enzyme.

There exists decades-old evidence that some mycobacteria, including Mycobacterium avium and Mycobacterium smegmatis, produce hydrazidase, an enzyme that can hydrolyze the first-line antitubercular agent isoniazid. Despite its importance as a potential resistance factor, no studies have attempted to reveal its identity. In this study, we aimed to isolate and identify M. smegmatis hydrazidase, characterize it, and evaluate its impact on isoniazid resistance. We determined the optimal condition under which M. smegmatis produced the highest amount of hydrazidase, purified the enzyme by column chromatography, and identified it by peptide mass fingerprinting. It was revealed to be PzaA, an enzyme known as pyrazinamidase/nicotinamidase whose physiological role remains unknown. The kinetic constants suggested that this amidase with broad substrate specificity prefers amides to hydrazides as a substrate. Notably, of the five tested compounds, including amides, only isoniazid served as an efficient inducer of pzaA transcription, as revealed by quantitative reverse transcription PCR. Moreover, high expression of PzaA was confirmed to be beneficial for the survival and growth of M. smegmatis in the presence of isoniazid. Thus, our findings suggest a possible role for PzaA, and other hydrazidases yet to be identified, as an intrinsic isoniazid resistance factor of mycobacteria.

A Culture Supernatant from an Actinomycete sp. Affects Biofilm Formation and Virulence Expression of Candida auris.

The multidrug-resistant pathogen Candida auris is characterized by its aggregation under certain conditions, which affects its biofilm formation, drug susceptibility, and pathogenicity. Although the innate tendency to aggregate depends on the strain, the mechanism regulating C. auris aggregation remains unclear. We found that the culture supernatant from one of the 95 Actinomyces strains isolated from a deep-sea environment (IMAs2016D-66) inhibited C. auris aggregation. The cells grown in the presence of IMAs2016D-66 exhibited reduced hydrophobicity, biofilm formation, and enhanced proteolytic activity. In addition, the efflux pump activity of the fluconazole-resistant C. auris strain LSEM 3673 was stimulated by IMAs2016D-66, whereas no significant change was observed in the fluconazole-susceptible strain LSEM 0643. As the relationship between aggregative tendency and virulence in C. auris is still unclear, IMAs2016D-66 can serve as a tool for investigating regulatory mechanisms of phenotype switching and virulence expression of C. auris. Understanding of phenotype switching may help us not only to understand the pathogenicity of C. auris, but also to design new drugs that target the molecules regulating virulence factors.

Discovery of a LuxR-type regulator involved in isoniazid-dependent gene regulation in Mycobacterium smegmatis.

OBJECTIVE: Most non-tuberculous mycobacteria exhibit intrinsic resistance against the anti-tuberculosis drug isoniazid (INH). We previously found that a pyrazinamidase/nicotinamidase of Mycobacterium smegmatis, named PzaA, has an enzymatic activity to hydrolyze INH, which may contribute to intrinsic resistance. Furthermore, PzaA expression is strongly induced by INH under nitrogen-depleted conditions, although the precise mechanism of this phenomenon remains unclear. Here, we aimed to reveal the mechanism underlying the INH-dependent induction of PzaA using a transcriptomic approach. METHODS: RNA sequencing was performed to identify INH-inducible genes other than pzaA. 5' rapid amplification of cDNA ends analysis was employed to identify the transcription start sites of INH-induced transcription units. The function of a LuxR-like regulator gene (MSMEI_1050) found within the gene cluster containing pzaA was confirmed by gene deletion and complementation experiments involving INH hydrolysis assay and quantitative reverse transcription PCR. RESULTS: RNA sequencing revealed 23 genes that INH strongly induced under conditions of nitrogen depletion, 17 of which were in a gene cluster containing pzaA. This cluster comprised at least three transcription units, including a non-INH-inducible monocistronic unit containing MSMEI_1050. Deletion of this gene deprived M. smegmatis of the ability to respond to INH, and complementation restored this ability. CONCLUSIONS: MSMEI_1050 plays a key role in INH-dependent gene regulation. The precise mechanism of action is to be determined in future studies.

Rifampicin exerts anti-mucoviscous activity against hypervirulent Klebsiella pneumoniae via binding to the RNA polymerase ß subunit.

OBJECTIVES: In hypervirulent Klebsiella pneumoniae (hvKP), the hypermucoviscous capsule is known to be a major virulence determinant. We previously discovered that rifampicin (RFP), a bactericidal drug that binds to and inhibits the ß subunit of RNA polymerase (RpoB), elicits anti-mucoviscous activity against hvKP by suppressing rmpA, a regulator of capsule production. Here, we aimed to determine whether RFP exerts this effect at sub-growth-inhibitory concentrations via its binding to RpoB. METHODS: Five spontaneous RFP-resistant mutants (R1-R5) were prepared from an hvKP clinical isolate and subjected to whole genome sequencing and mucoviscosity analyses. Subsequently, a two-step allelic exchange procedure was used to create a rpoB mutant R6 and revertants with wild-type rpoB from R1-R5 (named R1'-R5'). Transcription levels of rmpA and the capsular polysaccharide polymerase gene magA and capsule thickness of R1-R5 and R1'-R5' grown without or with RFP were evaluated by quantitative reverse transcription polymerase chain reaction and microscopic observation using India ink staining. RESULTS: R1-R5 all had non-synonymous point mutations in rpoB and were highly resistant to the bactericidal effects and anti-mucoviscous activity of RFP. While the properties of R6 were similar to those of R1-R5, the responses of R1'-R5' to RFP were identical to those of the wild type. rmpA and magA transcription levels and capsule thickness correlated well with the mucoviscosity levels. CONCLUSIONS: RFP exerts anti-mucoviscous activity by binding to RpoB. The mechanism of how this causes rmpA suppression remains to be explored.

A comprehensive list of the Bunyavirales replication promoters reveals a unique promoter structure in Nairoviridae differing from other virus families.

Members of the order Bunyavirales infect a wide variety of host species, including plants, animals and humans, and pose a threat to public health. Major families in this order have tri-segmented negative-sense RNA genomes, the 5' and 3' ends of which form complementary strands that serve as a replication promoter. Elucidation of the mechanisms by which viral polymerases recognize the promoter to initiate RNA synthesis is important for understanding viral replication and pathogenesis, and developing antivirals. A list of replication promoter configuration patterns may provide details on the differences in the replication mechanisms among bunyaviruses. By using public sequence data of all known bunyavirus species, we constructed a comprehensive list of the replication promoters comprising 40 nucleotides in both the 5' and 3' ends of the genome that form a specific complementary strand. Among tri-segmented bunyaviruses, members of the family Nairoviridae, including the highly pathogenic Crimean-Congo hemorrhagic fever virus, have evolved a GC-rich promoter structure differing from that of other families. The unique promoter structure might be related to the large genome size of the family Nairoviridae among bunyaviruses, and the large genome architecture might confer pathogenic advantages. The promoter list provided in this report is useful for predicting the virus family-specific replication mechanisms of bunyaviruses.

Genomic characterization of triple-carbapenemase-producing Acinetobacter baumannii.

OBJECTIVES: To characterize Acinetobacter baumannii OCU_Ac16a, a clinical isolate co-harbouring three acquired carbapenemase genes, bla NDM-1, bla TMB-1, and bla OXA-58, and assess the clinical significance of so-called multiple-carbapenemase producers. METHODS: OCU_Ac16a and its close relative, OCU_Ac16b, which lacks the bla NDM-1, were isolated from sputum cultures of a patient at Osaka City University Hospital. We subjected these strains to whole-genome analysis, particularly focusing on the genetic context of each carbapenemase gene. The transmissibility and functionality of each carbapenemase gene were analysed by conjugation and transformation experiments and antimicrobial susceptibility tests. RESULTS: bla TMB-1 was located in a class 1 integron on the chromosome, whereas bla NDM-1 and bla OXA-58 were found on plasmids named pOCU_Ac16a_2 and pOCU_Ac16a_3, respectively. pOCU_Ac16a_2 (which exhibited highly efficient self-transmissibility) and pOCU_Ac16a_3 (which did not show transmissibility but could be introduced into another A. baumannii strain via electroporation) could both confer carbapenem resistance (MICs ≥512 and ≥32 mg/L, respectively) on the recipient strain. The functionality of bla TMB-1 was evident from the high resistance of OCU_Ac16b to ceftazidime and cefepime (MICs ≥256 and 48 mg/L, respectively), and the high resistance of OCU_Ac16a to cefiderocol (MIC 32 mg/L) could be explained by the additive effect of bla NDM-1 and bla TMB-1. CONCLUSIONS: Our data revealed the genomic organization of OCU_Ac16a and demonstrated that all the carbapenemase genes are functional, each contributing to the extremely high broad-spectrum resistance of OCU_Ac16a to ß-lactams. As multiple-carbapenemase producers can be serious health threats as drug-resistant pathogens and disseminators of carbapenemase genes, close attention should be paid to their emergence.

Complete Genome Sequence of Acinetobacter pittii OCU_Ac17, Isolated from Human Venous Blood.

Acinetobacter pittii isolate OCU_Ac17 was obtained from the venous blood of a patient at a hospital in Japan. We present its complete 4.108-Mbp genome sequence (1 chromosome plus 3 plasmids), analyzed by combining long-read (Flongle) and short-read (MiniSeq) sequencing.

Complete Genome Sequence of a Clinical Isolate of Acinetobacter baumannii Harboring 11 Plasmids.

Here, we report the complete genome sequence of an Acinetobacter baumannii isolate harboring 11 plasmids, obtained at a hospital in Japan in 2016. The complete 4.07-Mbp genome sequence (1 chromosome and 11 plasmids) was analyzed by a combination of long-read (Flongle) and short-read (NovaSeq 6000) sequencing.

Clinical and virulence factors related to the 30-day mortality of Klebsiella pneumoniae bacteremia at a tertiary hospital: a case-control study.

Klebsiella pneumoniae bacteremia is a critical clinical presentation that is associated with high mortality. However, extremely few studies have investigated the virulence factors related to mortality of K. pneumoniae bacteremia in patients. The present study elucidated clinical and virulence factors associated with the 30-day mortality of K. pneumoniae bacteremia at a tertiary hospital. The medical records of 129 patients with K. pneumoniae bacteremia admitted to Osaka City University Hospital between January 2012 and December 2018 were retrospectively reviewed. Patient background characteristics, antimicrobial regimens, and prognosis were evaluated. Additionally, virulence factors were assessed using multiplex polymerase chain reaction to elucidate their association with K. pneumoniae. The 30-day mortality was 10.9% in patients with K. pneumoniae bacteremia. The male-to-female ratio, age, and underlying disease did not differ between the non-survivor and survivor groups. Multivariate analysis showed that sepsis (odds ratio (OR), 7.46; p = 0.005) and iutA (OR, 4.47; p = 0.046) were independent predictors associated with the 30-day mortality of K. pneumoniae bacteremia. Despite the relatively low 30-day mortality of patients with K. pneumoniae bacteremia, the treatment of those with sepsis and those infected with K. pneumoniae harboring iutA may require careful management for improving their outcomes.

Clinical characteristics of bacteremia caused by hypermucoviscous Klebsiella pneumoniae at a tertiary hospital.

This study aimed to assess the predictive factors of bacteremia due to hypermucoviscous Klebsiella pneumoniae (hvKP), as well as the mortality. The medical records of 114 patients with K. pneumoniae bacteremia who were divided into the hvKP (n = 24) and non-hvKP (n = 90) groups and were retrospectively reviewed. The male-to-female ratio, age, and underlying disease did not differ between the 2 groups. Mortality was higher among patients in the hvKP bacteremia group than in the non-hvKP bacteremia group (29.2% vs 6.7%). Multivariate analysis showed that the independent predictors associated with hvKP bacteremia were abscess (P = 0.01) and no antibiotic exposure (P = 0.02); thus, early assessment of these conditions is important. For patients with a history of abscess and no antibiotic exposure, it is necessary to administer treatment while keeping the risk of hvKP in mind.

Discovery of anti-mucoviscous activity of rifampicin and its potential as a candidate antivirulence agent against hypervirulent Klebsiella pneumoniae.

A recent increase in the incidence of hypervirulent Klebsiella pneumoniae (hvKP) infections, especially those caused by a sublineage of clonal group CG23 (CG23-I), is raising serious health concerns worldwide. The high virulence of hvKP is, at least in part, attributed to the overproduction of capsular polysaccharide (CPS), which is triggered by a positive regulator of capsular polysaccharide synthesis (cps) genes, named rmpA (regulator of mucoid phenotype A). Although extensive research has been conducted on the mechanisms of hvKP virulence, no study has focused on the development of antivirulence therapeutics. This study attempted to identify and validate an antimicrobial agent able to suppress hvKP hypermucoviscosity. A total of 18 commercially available antimicrobial agents, including ß-lactams, quinolones and aminoglycosides, were tested. Rifampicin (RFP) was found to have strong anti-mucoviscous activity against CG23-I hvKP even at subinhibitory concentrations. Polysaccharide extracts from hvKP showed substantially lowered viscosity when cells were grown with RFP. Moreover, microscopic observations demonstrated that RFP treatment results in a drastic reduction in the thickness of the CPS layer around hvKP cells. RFP treatment decreased transcript levels of rmpA and rmpA-regulated cps genes, indicating that RFP suppresses mucoviscosity of hvKP through inhibition of rmpA transcription. These data suggest that RFP may serve as a potential antivirulence agent for refractory hvKP infection.

Genome-Based Epidemiological Analysis of 13 Acinetobacter Strains Isolated from Blood Cultures of Hospitalized Patients from a University Hospital in Japan.

The genus Acinetobacter comprises many species that can cause infectious diseases. Despite their importance as nosocomial pathogens, the clinical distributions of individual species or clones are not well understood. In this study, we aimed to characterize 13 Acinetobacter strains isolated from blood cultures from Osaka City University Hospital. We conducted whole-genome sequencing to reveal their genetic background. We also performed PCR-based open reading frame typing (POT) and compared the results with those of multilocus sequence typing (MLST) to confirm its reliability as a genotyping method. Although biochemical analysis suggested that most isolates were A. baumannii, genomic analysis revealed that the collection of Acinetobacter isolates comprised six different species, with non-baumannii Acinetobacter species representing the majority. All strains possessed an inherent ADC-type ß-lactamase gene, whereas the distribution of OXA-type enzymes was limited to A. baumannii, A. pittii, and A. colistiniresistens. While MLST properly discriminated four A. baumannii strains as different clones, POT failed to distinguish three of the four A. baumannii strains from each other, highlighting a potential pitfall that may be encountered when applying POT to non-epidemiological A. baumannii strains.