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Abstract The increasing occurrence of hospital-associated infections, particularly bacteremia, caused by extensively drug-resistant (XDR) carbapenemase-producing colistin-resistant Klebsiella pneumoniae highlights a critical requirement to discover new therapeutic alternatives. Bacteriophages having host-specific bacteriolytic effects are promising alternatives for combating these pathogens. Among 12 phages isolated from public wastewater in Thailand, two phages-vB_kpnM_05 (myovirus) and vB_kpnP_08 (podovirus) showed broad-host range, producing bacteriolytic activities against 81.3% (n = 26) and 78.1% (n = 25) of 32 XDR carbapenemase-producing colistin-resistant K. pneumoniae, with capsular types—K15, K17, K50, K51, K52/wzi-50 and K2/wzi-2. Both phages showed short replication times, large burst sizes with rapid adsorptions. They exhibited significant stability under various environmental conditions. Genomic analysis revealed that both phages are genetically distinct phages from Myoviridae and Podoviridae family, with the lack of toxin, virulence, lysogeny and antibiotic resistance genes. These characteristics highlighted their promising potential for utilizing in phage therapy for combating XDR K. pneumoniae. Although phage cocktail combining vB_kpnM_05 and vB_kpnP_08 provided significant bacteriolysis for longer duration (8 h) than its monophage (6 h), bacterial regrowth was observed which suggested an evitable development of phage resistance under phages’ selection pressures. Future study will be undertaken to elucidate the precise mechanisms by which these XDR K. pneumoniae developed phage resistance and their associated fitness cost. Remarkably, combining phage cocktail with amikacin at their sub-inhibitory concentrations produced potent synergy by completely suppressing bacterial regrowth in vitro. Our study demonstrated the significant therapeutic and prophylactic effectiveness of a phage cocktail-amikacin combination as a promising alternative strategy for overcoming bacteremia associated with XDR K. pneumoniae having carbapenemase and colistin resistance in vivo.

Abstract Retrons are bacterial genetic elements that encode a reverse transcriptase and, in combination with toxic effector proteins, can serve as antiphage defense systems. However, the mechanisms of action of most retron effectors, and how phages evade retrons, are not well understood. Here, we show that some phages can evade retrons and other defense systems by producing specific tRNAs. We find that expression of retron-Eco7 effector proteins (PtuA and PtuB) leads to degradation of tRNATyr and abortive infection. The genomes of T5 phages that evade retron-Eco7 include a tRNA-rich region, including a highly expressed tRNATyr gene, which confers protection against retron-Eco7. Furthermore, we show that other phages (T1, T7) can use a similar strategy, expressing a tRNALys, to counteract a tRNA anticodon defense system (PrrC170).

Abstract Persistent bacterial lung infections in children lead to significant morbidity and mortality due to antibiotic resistance. In this paper, we describe how phage therapy has shown remarkable efficacy in preclinical and clinical studies, demonstrating significant therapeutic benefits through various administration routes. Ongoing trials are evaluating its safety and effectiveness against different pathogens. Advancing phage therapy through systematic studies and international collaboration could provide a viable alternative to traditional antibiotics for persistent infections.

Abstract Phages are gaining attention for their ability to target drug-resistant bacteria, disrupt biofilms, and reach intracellular pathogens, offering promising alternatives to traditional antibiotics. The Collection discusses advances in phage therapy, including their application in vaccine development, cancer immunotherapy, and gene delivery systems. Key research gaps are identified, such as challenges related to phage stability, immune response, and regulatory hurdles. Despite the progress, phage therapy faces obstacles in maintaining phage viability, evading immune detection, and navigating complex regulatory frameworks. The articles collectively address these challenges and propose potential solutions to enhance the effectiveness and acceptance of phage-based treatments. By overcoming these barriers, bacteriophage research has the potential to revolutionize medical therapies, providing innovative approaches to some of the most pressing healthcare challenges today.

Abstract In response to the escalating antibiotic resistance in multidrug-resistant pathogens, we propose an innovative phagemid-based capsid system to generate CRISPR-Cas13a-loaded antibacterial capsids (AB-capsids) for targeted therapy against multidrug-resistant Staphylococcus aureus. Our optimized phagemid system maximizes AB-capsid yield and purity, showing a positive correlation with phagemid copy number. Notably, an 8.65-fold increase in copy number results in a 2.54-fold rise in AB-capsid generation. Phagemids carrying terL-terS-rinA-rinB (prophage-encoded packaging site genes) consistently exhibit high packaging efficiency, and the generation of AB-capsids using lysogenized hosts with terL-terS deletion resulted in comparatively lower level of wild-type phage contamination, with minimal compromise on AB-capsid yield. These generated AB-capsids selectively eliminate S. aureus strains carrying the target gene while sparing non-target strains. In conclusion, our phagemid-based capsid system stands as a promising avenue for developing sequence-specific bactericidal agents, offering a streamlined approach to combat antibiotic-resistant pathogens within the constraints of efficient production and targeted efficacy.

Abstract In response to the escalating global threat of antimicrobial resistance, our laboratory has established a phagemid packaging system for the generation of CRISPR-Cas13a-antimicrobial capsids targeting methicillin-resistant Staphylococcus aureus (MRSA). However, a significant challenge arose during the packaging process: the unintentional production of wild-type phages alongside the antimicrobial capsids. To address this issue, the phagemid packaging system was optimized by strategically incorporated silent mutations. This approach effectively minimized contamination risks without compromising packaging efficiency. The study identified the indispensable role of phage packaging genes, particularly terL-terS, in efficient phagemid packaging. Additionally, the elimination of homologous sequences between the phagemid and wild-type phage genome was crucial in preventing wild-type phage contamination. The optimized phagemid-LSAB(mosaic) demonstrated sequence-specific killing, efficiently eliminating MRSA strains carrying target antibiotic-resistant genes. While acknowledging the need for further exploration across bacterial species and in vivo validation, this refined phagemid packaging system offers a valuable advancement in the development of CRISPR-Cas13a-based antimicrobials, shedding light on potential solutions in the ongoing battle against bacterial infections.

Abstract Escherichia coli O157 can cause foodborne outbreaks, with infection leading to severe disease such as hemolytic-uremic syndrome. Although phage-based detection methods for E. coli O157 are being explored, research on their specificity with clinical isolates is lacking. Here, we describe an in vitro assembly-based synthesis of vB_Eco4M-7, an O157 antigen-specific phage with a 68-kb genome, and its use as a proof of concept for E. coli O157 detection. Linking the detection tag to the C-terminus of the tail fiber protein, gp27 produces the greatest detection sensitivity of the 20 insertions sites tested. The constructed phage detects all 53 diverse clinical isolates of E. coli O157, clearly distinguishing them from 35 clinical isolates of non-O157 Shiga toxin-producing E. coli. Our efficient phage synthesis methods can be applied to other pathogenic bacteria for a variety of applications, including phage-based detection and phage therapy.

ABSTRACT The emergence of oxacillin-susceptible methicillin-resistant Staphylococcus aureus (OS-MRSA) has imposed further challenges to the clinical management of MRSA infections. When exposed to β-lactam antibiotics, these strains can easily acquire reduced β-lactam susceptibility through chromosomal mutations, including those in RNA polymerase (RNAP) genes such as rpoBC, which may then lead to treatment failure. Despite the increasing prevalence of such strains and the apparent challenges they pose for diagnosis and treatment, there is limited information available on the actual mechanisms underlying such chromosomal mutation-related transitions to reduced β-lactam susceptibility, as it does not directly associate with the expression of mecA. This study investigated the cellular physiology and metabolism of six missense mutants with reduced oxacillin susceptibility, each carrying respective mutations on RpoBH929P, RpoBQ645H, RpoCG950R, RpoCG498D, RpiAA64E, and FruBA211E, using capillary electrophoresis-mass spectrometry-based metabolomics analysis. Our results showed that rpoBC mutations caused RNAP transcription dysfunction, leading to an intracellular accumulation of ribonucleotides. These mutations also led to the accumulation of UDP-Glc/Gal and UDP-GlcNAc, which are precursors of UTP-associated peptidoglycan and wall teichoic acid. Excessive amounts of building blocks then contributed to the cell wall thickening of mutant strains, as observed in transmission electron microscopy, and ultimately resulted in decreased susceptibility to β-lactam in OS-MRSA.IMPORTANCEThe emergence of oxacillin-susceptible methicillin-resistant Staphylococcus aureus (OS-MRSA) strains has created new challenges for treating MRSA infections. These strains can become resistant to β-lactam antibiotics through chromosomal mutations, including those in the RNA polymerase (RNAP) genes such as rpoBC, leading to treatment failure. This study investigated the mechanisms underlying reduced β-lactam susceptibility in four rpoBC mutants of OS-MRSA. The results showed that rpoBC mutations caused RNAP transcription dysfunction, leading to an intracellular accumulation of ribonucleotides and precursors of peptidoglycan as well as wall teichoic acid. This, in turn, caused thickening of the cell wall and ultimately resulted in decreased susceptibility to β-lactam in OS-MRSA. These findings provide insights into the mechanisms of antibiotic resistance in OS-MRSA and highlight the importance of continued research in developing effective treatments to combat antibiotic resistance.

Phage therapy, the use of bacteriophages (phages) to treat bacterial infections, is regaining momentum as a promising weapon against the rising threat of multidrug-resistant (MDR) bacteria. This comprehensive review explores the historical context, the modern resurgence of phage therapy, and phage-facilitated advancements in medical and technological fields. It details the mechanisms of action and applications of phages in treating MDR bacterial infections, particularly those associated with biofilms and intracellular pathogens. The review further highlights innovative uses of phages in vaccine development, cancer therapy, and as gene delivery vectors. Despite its targeted and efficient approach, phage therapy faces challenges related to phage stability, immune response, and regulatory approval. By examining these areas in detail, this review underscores the immense potential and remaining hurdles in integrating phage-based therapies into modern medical practices.

Summary: This study investigated the potential of using SARS-CoV-2 viral concentrations in dust as an additional surveillance tool for early detection and monitoring of COVID-19 transmission. Dust samples were collected from 8 public locations in 16 districts of Bangkok, Thailand, from June to August 2021. SARS-CoV-2 RNA concentrations in dust were quantified, and their correlation with community case incidence was assessed. Our findings revealed a positive correlation between viral concentrations detected in dust and the relative risk of COVID-19. The highest risk was observed with no delay (0-day lag), and this risk gradually decreased as the lag time increased. We observed an overall decline in viral concentrations in public places during lockdown, closely associated with reduced human mobility. The effective reproduction number for COVID-19 transmission remained above one throughout the study period, suggesting that transmission may persist in locations beyond public areas even after the lockdown measures were in place.

The global increase in the prevalence of drug-resistant bacteria has necessitated the development of alternative treatments that do not rely on conventional antimicrobial agents. Using bacteriophage-derived lytic enzymes in antibacterial therapy shows promise; however, a thorough comparison and evaluation of their bactericidal efficacy are lacking. This study aimed to compare and investigate the bactericidal activity and spectrum of such lytic enzymes, with the goal of harnessing them for antibacterial therapy. First, we examined the bactericidal activity of spanins, endolysins, and holins derived from 2 Escherichia coli model phages, T1 and T7. Among these, T1-spanin exhibited the highest bactericidal activity against E. coli. Subsequently, we expressed T1-spanin within bacterial cells and assessed its bactericidal activity. T1-spanin showed potent bactericidal activity against all clinical isolates tested, including bacterial strains of 111 E. coli, 2 Acinetobacter spp., 3 Klebsiella spp., and 3 Pseudomonas aeruginosa. In contrast, T1 phage-derived endolysin showed bactericidal activity against E. coli and P. aeruginosa, yet its efficacy against other bacteria was inferior to that of T1-spanin. Finally, we developed a phage-based technology to introduce the T1-spanin gene into target bacteria. The synthesized non-proliferative phage exhibited strong antibacterial activity against the targeted bacteria. The potent bactericidal activity exhibited by spanins, combined with the novel phage synthetic technology, holds promise for the development of innovative antimicrobial agents.

Abstract Flavonoids are plant-produced secondary metabolites that are found ubiquitously. We have previously reported that apigenin, a class of flavonoid, has unique antimicrobial activity against Staphylococcus aureus (S. aureus), one of the major human pathogens. Apigenin inhibited fluoroquinolone-resistant S. aureus with DNA gyrase harboring the quinolone-resistant S84L mutation but did not inhibit wild-type DNA gyrase. In this study, we describe five flavonoids, quercetin, luteolin, kaempferol, baicalein, and commercially available CID12261165, that show similar antimicrobial activity against fluoroquinolone-resistant S. aureus. Among them, CID12261165 was the most effective with MIC values of ≤ 4 mg/L against quinolone-resistant S. aureus strains. In vitro DNA cleavage and supercoiling assays demonstrated inhibitory activity of CID12261165 against mutated DNA gyrase, whereas activity against wild-type DNA gyrase was not observed. CID12261165 also inhibited quinolone-resistant Enterococci with an MIC value of 8 mg/L. While fluoroquinolone-resistant amino acid replacements can improve the fitness of bacterial cells, it is unknown why quinolone-susceptible S. aureus strains were predominant before the introduction of fluoroquinolone. The present study discusses the current discrepancies in the interpretation of antimicrobial activities of flavonoids, as well as the possible reasons for the preservation of wild-type DNA gyrase wherein the environmental flavonoids cannot be ignored.

Abstract Staphylococcus virus ΦSA012 has a wide host range and efficient lytic activity. Here, we assessed the biological stability of ΦSA012 against temperature, freeze-thawing, and pH to clinically apply the phage. In addition, inoculation of ΦSA012 through i.p. and i.v. injections into mice revealed that phages were reached the limit of detection in serum and accumulated notably spleens without inflammation at 48 h post-inoculation. Furthermore, inoculation of ΦSA012 through s.c. injections in mice significantly induced IgG, which possesses neutralizing activity against ΦSA012 and other Staphylococcus viruses, ΦSA039 and ΦMR003, but not Pseudomonas viruses ΦS12-3 and ΦR18 or Escherichia viruses T1, T4, and T7 in vitro. Immunoelectron microscopic analysis showed that purified anti-phage IgG recognizes the long-tail fiber of staphylococcus viruses. Although S. aureus inoculation resulted in a 25% survival rate in a mouse i.p. model, ΦSA012 inoculation (i.p.) improved the survival rate to 75%; however, the survival rate of ΦSA012-immunized mice decreased to less than non-immunized mice with phage i.v. injection at a MOI of 100. These results indicated that ΦSA012 possesses promise for use against staphylococcal infections but we should carefully address the appropriate dose and periods of phage administration. Our findings facilitate understandings of staphylococcus viruses for phage therapy.

Summary: Developing an effective therapy to overcome carbapenemase-positive Klebsiella pneumoniae (CPKp) is an important therapeutic challenge that must be addressed urgently. Here, we explored a Ca-EDTA combination with aztreonam or ceftazidime-avibactam in vitro and in vivo against diverse CPKp clinical isolates. The synergy testing of this study demonstrated that novel aztreonam-Ca-EDTA or ceftazidime-avibactam-Ca-EDTA combination was significantly effective in eliminating planktonic and mature biofilms in vitro, as well as eradicating CPKp infections in vivo. Both combinations revealed significant therapeutic efficacies in reducing bacterial load in internal organs and protecting treated mice from mortality. Conclusively, this is the first in vitro and in vivo study to demonstrate that novel aztreonam-Ca-EDTA or ceftazidime-avibactam-Ca-EDTA combinations provide favorable efficacy and safety for successful eradication of carbapenemase-producing Klebsiella pneumoniae planktonic and biofilm infections.

An automated amplification-free digital RNA detection platform using CRISPR-Cas13a and magnetic bead technology implemented in a microchamber device demonstrates sensitive SARS-CoV-2 genomic RNA detection and variant discrimination.

Bacteriophages, the viruses that infect and replicate within bacteria, have long been recognized as potential therapeutic agents against bacterial infections [...]

Abstract Previous studies indicated residents in geriatric long-term care facilities (LTCFs) had much higher prevalence of extended-spectrum β-lactamase-producing Enterobacteriaceae (ESBL-E) carriage than the general population. Most ESBL-E carriers are asymptomatic. The study tested the hypothesis that residents with ESBL-E carriage may accumulate inside geriatric LTCFs through potential cross-transmission after exposure to residents with prolonged ESBL-E carriage. 260 residents from four Japanese LTCFs underwent ESBL-E testing of fecal specimens and were divided into two cohorts: Cohort 1,75 patients with ≥ 2 months residence at study onset; Cohort 2, 185 patients with

Abstract Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems are one of the factors which can contribute to limiting the development and evolution of antibiotic resistance in bacteria. There are three genomic loci of CRISPR-Cas in Enterococcus faecalis. In this study, we aimed to assess correlation of the CRISPR-Cas system distribution with the acquisition of antibiotic resistance among E. faecalis isolates. A total of 151 isolates of E. faecalis were collected from urinary tract infections (UTI) and dental-root canal (DRC). All isolates were screened for phenotypic antibiotic resistance. In addition, antibiotic resistance genes and CRISPR loci were screened by using polymerase chain reaction. Genomic background of the isolates was identified by random amplified polymorphic DNA (RAPD)-PCR. The number of multidrug-resistant E. faecalis strains were higher in UTI isolates than in DRC isolates. RAPD-PCR confirmed that genomic background was diverse in UTI and DRC isolates used in this study. CRISPR loci were highly accumulated in gentamycin-, teicoplanin-, erythromycin-, and tetracycline-susceptible strains. In concordance with drug susceptibility, smaller number of CRISPR loci were identified in vanA, tetM, ermB, aac6’-aph(2”), aadE, and ant(6) positive strains. These data indicate a negative correlation between CRISPR-cas loci and antibiotic resistance, as well as, carriage of antibiotic resistant genes in both of UTI and DRC isolates.

Abstract Background A high prevalence of methicillin-resistant Staphylococcus aureus (MRSA) colonization has been reported among residents in geriatric long-term care facilities (LTCFs). Some studies indicate that MRSA might be imported from hospitals into LTCFs via resident transfer; however, other studies report that high MRSA prevalence might be caused by cross-transmission inside LTCFs. We aimed to assess which factors have a large impact on the high MRSA prevalence among residents of geriatric LTCFs. Methods We conducted a cohort study among 260 residents of four geriatric LTCFs in Japan. Dividing participants into two cohorts, we separately analyzed (1) the association between prevalence of MRSA carriage and length of LTCF residence (Cohort 1: n = 204), and (2) proportion of residents identified as MRSA negative who were initially tested at admission but subsequently identified as positive in secondary testing performed at ≥2 months after their initial test (Cohort 2: n = 79). Results Among 204 residents in Cohort 1, 20 (9.8%) were identified as positive for MRSA. Compared with residents identified as MRSA negative, a larger proportion of MRSA-positive residents had shorter periods of residence from the initial admission (median length of residence: 5.5 vs. 2.8 months), although this difference was not statistically significant (p = 0.084). Among 79 residents in Cohort 2, 60 (75.9%) were identified as MRSA negative at the initial testing. Of these 60 residents, only one (1.7%) had subsequent positive conversion in secondary MRSA testing. In contrast, among 19 residents identified as MRSA positive in the initial testing, 10 (52.6%) were negative in secondary testing. Conclusions The prevalence of MRSA was lower among residents with longer periods of LTCF residence than among those with shorter periods. Furthermore, few residents were found to become MRSA carrier after their initial admission. These findings highlight that MRSA in LTCFs might be associated with resident transfer rather than spread via cross-transmission inside LTCFs.

CRISPR technology is emerging as a potential antimicrobial against antimicrobial-resistant bacteria. Here the authors develop a bacteriophage delivered Cas13a system for killing target bacteria and detecting bacterial genes.

Staphylococcus spp. colonize commensally on the human skin. Some commensal coagulase-negative staphylococci and Staphylococcus aureus are also involved in nosocomial infections. Bacteria were collected from skin healed from pressure injury (PI). After the collection time points, some patients suffered from recurrent PI (RPI). This study analyzed the characteristics of Staphylococcus spp. on healed skin before recurrence between healed skin that suffered from RPI within 6 weeks (RPI group) and healed skin that did not suffer within the duration (non-RPI group) by Staphylococcus spp.-specific sequencing. Of the seven patients in the RPI group, two were dominated by S. aureus and four by Staphylococcus caprae, coagulase-negative human commensal staphylococci in the RPI group. Using mouse models, both S. caprae and S. aureus, but not Staphylococcus epidermidis, colonized on skin healed from injury at significantly higher rates than normal skin. Although subcutaneous injection of S. caprae did not induce lesion formation, the bacterium exhibited high hemolytic activity on human red blood cells. Lesion formation by subcutaneous injection of S. aureus was significantly suppressed in the presence of S. caprae. The hemolytic activity of rabbit blood cells of S. aureus was suppressed by S. caprae, whereas the hemolytic activity of S. caprae was dramatically suppressed by S. aureus. Data indicated that each of the two Staphylococcus spp. suppresses the pathogenicity of the other and that the imbalance between the two is associated with RPI.

Non-menstrual toxic shock syndrome (non-mTSS) is a life-threatening disease caused by Staphylococcus aureus strains producing superantigens, such as staphylococcal enterotoxins A, B, C, and toxic shock syndrome toxin-1 (TSST-1). However, little is known about why the TSS cases are rare, although S. aureus strains frequently carry a tst gene, which encodes TSST-1. To answer this question, the amount of TSST-1 produced by 541 clinical isolates was measured in both the presence and absence of serum supplementation to growth media. Then a set of S. aureus strains with similar genetic backgrounds isolated from patients presenting with non-mTSS and those with clinical manifestations other than non-mTSS was compared for their TSST-1 inducibility by human serum, and their whole-genome sequences were determined. Subsequently, the association of mutations identified in the tst promoter of non-mTSS strains with TSST-1 inducibility by human serum was evaluated by constructing promoter replacement mutants and green fluorescent protein (GFP) reporter recombinants. Results showed that 39 out of 541 clinical isolates (7.2%), including strains isolated from non-mTSS patients, had enhanced production of TSST-1 in the presence of serum. TSST-1 inducibility by human serum was more clearly seen in non-mTSS strains of clonal complex (CC)-5. Moreover, the whole-genome sequence analysis identified a set of sequence variations at a putative SarA-binding site of the tst promoter. This sequence variation was proven to be partially responsible for the induction of TSST-1 production by human serum. We conclude that the onset of staphylococcal toxic shock syndrome caused by TSST-1-producing CC-5 strains seem at least partially initiated by serum induction of TSST-1, which is regulated by the mutation of putative SarA-binding site at the tst promoter.

We present a case of life-threatening pneumonia caused by Pseudomonas aeruginosa (P. aeruginosa) in a healthy 67-year-old man. Rapid disseminated infection resulted in the right hemorrhagic pneumonia and bacteremia. Antimicrobial therapy had limited effects, radical pneumonectomy eventually resolved the prolonged infection. Concurrently, we explored the environmental factors responsible for fulminant P. aeruginosa infection. Multi-locus sequence typing demonstrated that P. aeruginosa isolated from the patient was identical to that collected from home whirlpool bath by the common virulent factor gene. Massive inhalation of contaminated aerosol and pathogen virulence may have synergistically contributed to the severity in this case.

Abstract Background Staphylococcus caprae is an animal-associated bacterium regarded as part of goats’ microflora. Recently, S. caprae has been reported to cause human nosocomial infections such as bacteremia and bone and joint infections. However, the mechanisms responsible for the development of nosocomial infections remain largely unknown. Moreover, the complete genome sequence of S. caprae has not been determined. Results We determined the complete genome sequences of three methicillin-resistant S. caprae strains isolated from humans and compared these sequences with the genomes of S. epidermidis and S. capitis, both of which are closely related to S. caprae and are inhabitants of human skin capable of causing opportunistic infections. The genomes showed that S. caprae JMUB145, JMUB590, and JMUB898 strains contained circular chromosomes of 2,618,380, 2,629,173, and 2,598,513 bp, respectively. JMUB145 carried type V SCCmec, while JMUB590 and JMUB898 had type IVa SCCmec. A genome-wide phylogenetic SNP tree constructed using 83 complete genome sequences of 24 Staphylococcus species and 2 S. caprae draft genome sequences confirmed that S. caprae is most closely related to S. epidermidis and S. capitis. Comparative complete genome analysis of eight S. epidermidis, three S. capitis and three S. caprae strains revealed that they shared similar virulence factors represented by biofilm formation genes. These factors include wall teichoic acid synthesis genes, poly-gamma-DL-glutamic acid capsule synthesis genes, and other genes encoding nonproteinaceous adhesins. The 17 proteinases/adhesins and extracellular proteins known to be associated with biofilm formation in S. epidermidis were also conserved in these three species, and their biofilm formation could be detected in vitro. Moreover, two virulence-associated gene clusters, the type VII secretion system and capsular polysaccharide biosynthesis gene clusters, identified in S. aureus were present in S. caprae but not in S. epidermidis and S. capitis genomes. Conclusion The complete genome sequences of three methicillin-resistant S. caprae isolates from humans were determined for the first time. Comparative genome analysis revealed that S. caprae is closely related to S. epidermidis and S. capitis at the species level, especially in the ability to form biofilms, which may lead to increased virulence during the development of S. caprae infections.

A high prevalence of extended-spectrum β-lactamase-producing Enterobacterales (ESBL-PE) may call for monitoring in geriatric long-term care facilities (g-LTCFs). We surveyed the distribution of ESBL-causative gene types and antimicrobial susceptibility in ESBL-PE strains from residents in g-LTCFs, and investigated the association between ESBL-causative gene types and antimicrobial susceptibility. First, we analyzed the types of ESBL-causative genes obtained from 141 ESBL-PE strains collected from the feces of residents in four Japanese g-LTCFs. Next, we determined the minimum inhibitory concentration values for alternative antimicrobial agents against ESBL-PE, including β-lactams and non-β-lactams. Escherichia coli accounted for 96% of the total ESBL-PE strains. Most strains (94%) contained blaCTX-M group genes. The genes most commonly underlying resistance were of the blaCTX-M-9 and blaCTX-M-1 groups. Little difference was found in the distribution of ESBL-causative genes among the facilities; however, antimicrobial susceptibility differed widely among the facilities. No specific difference was found between antimicrobial susceptibility and the number of ESBL-causative genes. Our data showed that ESBL-PEs were susceptible to some antimicrobial agents, but the susceptibility largely differed among facilities. These findings suggest that each g-LTCF may require specific treatment strategies based on their own antibiogram. Investigations into drug resistance should be performed in g-LTCFs as well as acute medical facilities.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas13a, previously known as CRISPR-C2c2, is the most recently identified RNA-guided RNA-targeting CRISPR-Cas system that has the unique characteristics of both targeted and collateral single-stranded RNA (ssRNA) cleavage activities. This system was first identified in Leptotrichia shahii. Here, the complete whole genome sequences of 11 Leptotrichia strains were determined and compared with 18 publicly available Leptotrichia genomes in regard to the composition, occurrence and diversity of the CRISPR-Cas13a, and other CRISPR-Cas systems. Various types of CRISPR-Cas systems were found to be unevenly distributed among the Leptotrichia genomes, including types I-B (10/29, 34.4%), II-C (1/29, 2.6%), III-A (6/29, 15.4%), III-D (6/29, 15.4%), III-like (3/29, 7.7%), and VI-A (11/29, 37.9%), while 8 strains (20.5%) had no CRISPR-Cas system at all. The Cas13a effectors were found to be highly divergent with amino acid sequence similarities ranging from 61% to 90% to that of L. shahii, but their collateral ssRNA cleavage activities leading to impediment of bacterial growth were conserved. CRISPR-Cas spacers represent a sequential achievement of former intruder encounters, and the retained spacers reflect the evolutionary phylogeny or relatedness of strains. Analysis of spacer contents and numbers among Leptotrichia species showed considerable diversity with only 4.4% of spacers (40/889) were shared by two strains. The organization and distribution of CRISPR-Cas systems (type I-VI) encoded by all registered Leptotrichia species revealed that effector or spacer sequences of the CRISPR-Cas systems were very divergent, and the prevalence of types I, III, and VI was almost equal. There was only one strain carrying type II, while none carried type IV or V. These results provide new insights into the characteristics and divergences of CRISPR-Cas systems among Leptotrichia species.

The bacteriophage (or phage for short) has been used as an antibacterial agent for over a century but was abandoned in most countries after the discovery and broad use of antibiotics. The worldwide emergence and high prevalence of antimicrobial-resistant (AMR) bacteria have led to a revival of interest in the long-forgotten antibacterial therapy with phages (phage therapy) as an alternative approach to combatting AMR bacteria. The rapid progress recently made in molecular biology and genetic engineering has accelerated the generation of phage-related products with superior therapeutic potentials against bacterial infection. Nowadays, phage-based technology has been developed for many purposes, including those beyond the framework of antibacterial treatment, such as to suppress viruses by phages, gene therapy, vaccine development, etc. Here, we highlighted the current progress in phage engineering technology and its application in modern medicine.

Differences in the gut microbial content of Lutzomyia (Lu.) ayacuchensis, a primary vector of Andean-type cutaneous leishmaniasis in Ecuador and Peru, may influence the susceptibility of these sand flies to infection by Leishmania. As a first step toward addressing this hypothesis, a comparative analysis of bacterial and fungal compositions from Lu. ayacuchensis populations with differential susceptibilities to Leishmania was performed. Bacterial 16S rRNA gene amplification and Illumina MiSeq sequencing approaches were used to characterize the bacterial composition in wild-caught populations from the Andean areas of Ecuador and southern Peru at which the sand fly species transmit Leishmania (Leishmania) mexicana and Leishmania (Viannia) peruviana, respectively, and a population from the northern Peruvian Andes at which the transmission of Leishmania by Lu. ayacuchensis has not been reported. In the present study, 59 genera were identified, 21 of which were widely identified and comprised more than 95% of all bacteria. Of the 21 dominant bacterial genera identified in the sand flies collected, 10 genera had never been detected in field sand flies. The Ecuador and southern Peru populations each comprised individuals of particular genera, while overlap was clearly observed between microbes isolated from different sites, such as the number of soil organisms. Similarly, Corynebacterium and Micrococcus were slightly more dominant bacterial genera in the southern Peru population, while Ochrobactrum was the most frequently isolated from other populations. On the other hand, fungi were only found in the southern Peru population and dominated by the Papiliotrema genus. These results suggest that variation in the insect gut microbiota may be elucidated by the ecological diversity of sand flies in Peru and Ecuador, which may influence susceptibility to Leishmania infection. The present study provides key insights for understanding the role of the microbiota during the course of L. (L.) mexicana and L. (V.) peruviana infections in this important vector.

Klebsiella variicola, a member of K. pneumoniae phylogroup, can cause severe infectious diseases. We report a case of K. variicola meningitis after neurosurgery. The bacterium was isolated from blood and cerebrospinal fluid, and bacterial species identification was carried out by using both matrix-assisted laser-desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF MS) and whole genome sequencing. Initially, the organism was misidentified as K. pneumoniae by VITEK®2; automated system in the clinical laboratory examination. The patient recovered with the combination of surgical drainage and antimicrobial treatment. To our knowledge, this is the first case report of post-surgical meningitis caused by K. variicola. As experienced in this case, the automated bacterial identification system popularly being used in the clinical laboratory might not be effective enough for bacterial species identification. The use of MALDI-TOF MS for microbial identification may be helpful to physicians for appropriate management of K. pneumoniae phylogroup infection. Keywords: Klebsiella variicola, Post-surgical meningitis, MALDI-TOF MS

Increased intestinal permeability is thought to underlie the pathogenesis of food allergy. We explore the mechanism responsible for changes in the morphology and function of the intestinal barrier using a rat model of food allergy, focusing on the contribution of intestinal microbiota. Juvenile−young adult rats were sensitized with ovalbumin and treated with antibiotics or probiotics (Clostridium butyricum and Lactobacillus reuteri), respectively. The serum ovalbumin-IgE levels, intestinal permeability, histopathological features, tight junction (TJ)-associated proteins, Th2 cytokines, and gut microbiota in feces were analyzed in each group. Sensitized rats showed an increase in ovalbumin-IgE levels and intestinal permeability with gut mucosal inflammation, whereas rats that received probiotics were only mildly affected. Rats given ovalbumin, but not those given probiotics, showed a reduction in both TJ-related protein expression and localization. Th2 cytokine levels were increased in the sensitized rats, but not in those given probiotics. TJs in rats treated with ovalbumin and antibiotics were disrupted, but those in rats administered probiotics were undamaged. Clostridiaceae were increased in the probiotics groups, especially Alkaliphilus, relative to the ovalbumin-sensitized group. Gut microbiota appears to play a role in regulating epithelial barrier function, and probiotics may help to prevent food sensitization through the up-regulation of TJ proteins.