Association of mprF mutations with cross-resistance to daptomycin and vancomycin in methicillin-resistant Staphylococcus aureus (MRSA).

We first reported a phenomenon of cross-resistance to vancomycin (VCM) and daptomycin (DAP) in methicillin-resistant Staphylococcus aureus (MRSA) in 2006, but mechanisms underlying the cross-resistance remain incompletely understood. Here, we present a follow-up study aimed to investigate genetic determinants associated with the cross-resistance. Using 12 sets of paired DAP susceptible (DAPS) and DAP non-susceptible (DAPR) MRSA isolates from 12 patients who had DAP therapy, we (i) assessed susceptibility to DAP and VCM, (ii) compared whole-genome sequences, (iii) identified mutations associated with cross-resistance to DAP and VCM, and (iv) investigated the impact of altered gene expression and metabolic pathway relevant to the cross-resistance. We found that all 12 DAPR strains exhibiting cross-resistance to DAP and VCM carried mutations in mprF, while one DAPR strain with reduced susceptibility to only DAP carried a lacF mutation. On the other hand, among the 32 vancomycin-intermediate S. aureus (VISA) strains isolated from patients treated with VCM, five out of the 18 strains showing cross-resistance to DAP and VCM carried a mprF mutation, while 14 strains resistant to only VCM had no mprF mutation. Moreover, substitution of mprF in a DAPS strain with mutated mprF resulted in cross-resistance and vice versa. The elevated lysyl-phosphatidylglycerol (L-PG) production, increased positive bacterial surface charges and activated cell wall (CW) synthetic pathways were commonly found in both clinical isolates and laboratory-developed mutants that carry mprF mutations. We conclude that mprF mutation is responsible for the cross-resistance of MRSA to DAP and VCM, and treatment with DAP is more likely to select for mprF-mediated cross-resistance than is with VCM.

Identification and characterization of mutations responsible for the ß-lactam resistance in oxacillin-susceptible mecA-positive Staphylococcus aureus.

Staphylococcus aureus strains that are susceptible to the ß-lactam antibiotic oxacillin despite carrying mecA (OS-MRSA) cause serious clinical problems globally because of their ability to easily acquire ß-lactam resistance. Understanding the genetic mechanism(s) of acquisition of the resistance is therefore crucial for infection control management. For this purpose, a whole-genome sequencing-based analysis was performed using 43 clinical OS-MRSA strains and 100 mutants with reduced susceptibility to oxacillin (MICs 1.0-256 µg/mL) generated from 26 representative OS-MRSA strains. Genome comparison between the mutants and their respective parent strains identified a total of 141 mutations in 46 genes and 8 intergenic regions. Among them, the mutations are frequently found in genes related to RNA polymerase (rpoBC), purine biosynthesis (guaA, prs, hprT), (p)ppGpp synthesis (relSau), glycolysis (pykA, fbaA, fruB), protein quality control (clpXP, ftsH), and tRNA synthase (lysS, gltX), whereas no mutations existed in mec and bla operons. Whole-genome transcriptional profile of the resistant mutants demonstrated that expression of genes associated with purine biosynthesis, protein quality control, and tRNA synthesis were significantly inhibited similar to the massive transcription downregulation seen in S. aureus during the stringent response, while the levels of mecA expression and PBP2a production were varied. We conclude that a combination effect of mecA upregulation and stringent-like response may play an important role in acquisition of ß-lactam resistance in OS-MRSA.

Development of CRISPR-Cas13a-based antimicrobials capable of sequence-specific killing of target bacteria.

The emergence of antimicrobial-resistant bacteria is an increasingly serious threat to global health, necessitating the development of innovative antimicrobials. Here we report the development of a series of CRISPR-Cas13a-based antibacterial nucleocapsids, termed CapsidCas13a(s), capable of sequence-specific killing of carbapenem-resistant Escherichia coli and methicillin-resistant Staphylococcus aureus by recognizing corresponding antimicrobial resistance genes. CapsidCas13a constructs are generated by packaging programmed CRISPR-Cas13a into a bacteriophage capsid to target antimicrobial resistance genes. Contrary to Cas9-based antimicrobials that lack bacterial killing capacity when the target genes are located on a plasmid, the CapsidCas13a(s) exhibit strong bacterial killing activities upon recognizing target genes regardless of their location. Moreover, we also demonstrate that the CapsidCas13a(s) can be applied to detect bacterial genes through gene-specific depletion of bacteria without employing nucleic acid manipulation and optical visualization devices. Our data underscore the potential of CapsidCas13a(s) as both therapeutic agents against antimicrobial-resistant bacteria and nonchemical agents for detection of bacterial genes.

Complete Genome Sequence of a Panton-Valentine Leukocidin-Negative Staphylococcus aureus Strain Isolated from a Patient with Pervasive Necrotizing Soft Tissue Infection.

The association of Panton-Valentine leukocidin (PVL) toxin with necrotizing soft tissue infection (NSTI) caused by Staphylococcus aureus remains controversial. Here, we report the complete genome sequence of the PVL-negative S. aureus strain JMUB1273, isolated from a patient with pervasive NSTI.

Corrigendum: Composition and Diversity of CRISPR-Cas13a Systems in the Genus Leptotrichia.

[This corrects the article DOI: 10.3389/fmicb.2019.02838.].

Complete Genome Sequence of the Methicillin-Resistant Staphylococcus aureus Strain JMUB3031, Isolated from a Patient with Fatal Community-Acquired Pneumonia.

Severe community-acquired pneumonia (CAP) caused by methicillin-resistant Staphylococcus aureus (MRSA) is relatively rare and is usually associated with rapid progression to death. Here, we report the complete genome sequence of the MRSA strain JMUB3031, which was isolated from a patient with fatal CAP.

Composition and Diversity of CRISPR-Cas13a Systems in the Genus Leptotrichia.

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.

An MMP/TIMP ratio scoring system as a potential predictive marker of diabetic foot ulcer healing.

OBJECTIVES: The mechanism of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in diabetic foot ulcers (DFUs) is unclear. The purpose of this study was to describe changes in MMP-1, MMP-9, and TIMP-1 levels during DFU healing, and to search for any correlation in the changes in MMP levels with wound healing, in order to find possible predictors of healing. METHODS: Patients with a DFU were recruited and placed into two groups, according to the degree of wound healing: 'good healers' and 'poor healers'. Levels of MMP-1, MMP-9, and TIMP-1 were analysed by ELISA (enzyme-linked immunosorbent assay). RESULTS: A total of 22 patients participated in the study. The MMP-1 level was significantly higher at weeks zero (W0) and 12 (W12) in 'good healers' than in 'poor healers' (p=0.045 and 0.008, respectively). In contrast, the MMP-9 level was significantly lower in 'good healers' than in 'poor healers' at W0, W4, and W12 (p=0.001, 0.001 and 0.028, respectively). Receiver operator curve (ROC) analysis of the MMP-9 level, MMP-1/TIMP-1 ratio, and MMP-9/TIMP-1 ratio at W0 provided cut-off levels of 0.38, 0.056, and 9.06, respectively, which were best predictive of a reduction in wound area at W4 ('good healers' versus 'poor healers'; thereby predicting wound healing condition at W12) with a sensitivity of 81.8%, 81.8%, and 90.9%, and a specificity of 64.6%, 55%, and 64.6%, respectively. CONCLUSION: A 'poor healing scoring system' is therefore proposed that could be determined on patient admission, which has the potential to be used clinically as a predictor of healing, thus allowing an appropriate treatment plan to be developed.

Complete genome sequencing of three human clinical isolates of Staphylococcus caprae reveals virulence factors similar to those of S. epidermidis and S. capitis.

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.

Wound Fluid Matrix Metalloproteinase-9 as a Potential Predictive Marker for the Poor Healing Outcome in Diabetic Foot Ulcers.

BACKGROUND AND OBJECTIVE: Evidence for the roles of matrix metalloproteinases-9 (MMP-9) in the healing process of diabetic foot ulcers has remained unclear. We therefore aimed to demonstrate the relationship of MMP-9 with the wound healing process and determine its potential usefulness in predicting the wound healing outcome. METHODS: Twenty-two patients with diabetic foot ulcer were recruited. The wound size was determined, and the wound fluid was collected for the measurement of MMP-9 levels using an ELISA during the 12-week follow-up period regularly. The patients were categorized as good healers and poor healers when the wound area reduction was ≥ 50% and < 50% at week 4 when compared to the initial wound size at week 0. RESULTS: Median wound fluid MMP-9 levels in the poor healer group were shown to be significantly higher than those in the good healer group (1.03 pg/µg protein vs. 0.06 pg/µg protein, p = 0.001), and the levels fluctuated throughout the 12-week follow-up period. In contrast to the poor healer group, the MMP-9 levels were demonstrated to be constantly low throughout the follow-up period in the good healer group. ROC analysis showed that the MMP-9 level of 0.38 pg/µg protein was able to predict the wound healing outcome with the sensitivity of 81.8%, the specificity of 64.6%, and the area under the curve of 0.901 (CI 0.78-1.03, p = 0.001). CONCLUSION: These findings suggested that determination of wound fluid MMP-9 levels might become a promising biomarker predicting wound healing outcomes and a novel potential therapeutic target for diabetic foot ulcers.

Prevalence and hematological parameters of thalassemia in Tha Kradarn subdistrict Chachoengsao Province, Thailand.

OBJECTIVE: To determine the prevalence, molecular characteristics and hematological study of thalassemia in Tha Kradarn Subdistrict Chachoengsao Province. MATERIAL AND METHOD: The present study population consisted of266 participants from Moo 19 Baan Na-Ngam, Chachoengsao Province, Thailand. After blood collection, all samples were screened for thalassemia by initial screening with the OF and DCIP tests and additional testing by CBC, RBC indices, hemoglobin typing and determination of Hb A2 and Hb E. All common alpha-thalassemia mutations were determined using the PCR with allele specific primers and Gap PCR for common deletions. RESULTS: The prevalence of alpha-thal 1, alpha-thal 2 and beta-thal were found as 2.72%, 11.26% and 0.97%, respectively. Regarding the abnormal hemoglobins, the prevalence of Hb E, Hb Constant Spring and Hb Pakse was 38.45%, 3.69% and 0.78%, respectively. MCV and MCH were significantly different between P-thalassemia as well as a-thal 1 carriers and normal subjects. In all alpha-thal 1 traits, it was found that the MCV and MCH were less than 75 fL and 25 pg, therefore, these parameters can be used for alpha-thal 1 screening. CONCLUSION: In the present study, the prevalence of thalassemia was similar to previous studies. Moreover, using the combination of OF and DCIP tests compared with MCV, MCH and DCIP tests for the initial thalassemia screening, it was found that the OF and DCIP tests gave more false positive results, which increased the need for further Hb typing. Hence, the MCV and MCH combined with DCIP tests provide cost minimization and practical for a large population-based screening program.

Screening for thalassemia and hemoglobinopathy in a rural area of Thailand: a preliminary study.

The present study aimed to screen thalassemia and hemoglobinopathy in Baan Na-Ngam, Chachoengsao Province, Thailand. Blood samples were obtained from 266 volunteers; 105 males and 161 females aged 7 to 49 years. Blood samples screened for thalassemia combining the OF and modified DCIP precipitation tests. CBC, RBC indices, hemoglobin typing, HbA2 and Hb E were determined. Combined OF and DCIP tests found that in normal subjects, 128 out of 155 were negative for both, 3 were -/+ pattern, 22 were +/- pattern and 2 was positive for both. Interestingly, one sample showed an abnormal hemoglobin pattern, which could not be determined by automated LPLC. Three beta-thalassemia trait subjects were positive for only the OF test. For the Hb E trait, 57 out of 94 were -/+ pattern; 37 were positive for both tests. Moreover, 14 homozygous Hb E subjects were positive for both tests. The prevalence of beta-thalassemia trait was 1.1%, Hb E trait was 35.3% and homozygous Hb E was 5.3%. Since DNA analysis was not performed, alpha-thalassemia1 and alpha-thalassemia2 traits cannot be excluded. In conclusion, a combination of the OF and DCIP tests is suitable for preliminary screening for thalassemia and hemoglobinopathy. However, RBC parameters, hemoglobin typing and PCR analysis will provide more specific diagnosis, especially in alpha-thalassemias.