Comparative Study of Two-Dimensional (2D) vs. Three-Dimensional (3D) Organotypic Kertatinocyte-Fibroblast Skin Models for Staphylococcus aureus (MRSA) Infection.

The invasion of skin tissue by Staphylococcus aureus is mediated by mechanisms that involve sequential breaching of the different stratified layers of the epidermis. Induction of cell death in keratinocytes is a measure of virulence and plays a crucial role in the infection progression. We established a 3D-organotypic keratinocyte-fibroblast co-culture model to evaluate whether a 3D-skin model is more effective in elucidating the differences in the induction of cell death by Methicillin-resistant Staphylococcus aureus (MRSA) than in comparison to 2D-HaCaT monolayers. We investigated the difference in adhesion, internalization, and the apoptotic index in HaCaT monolayers and our 3D-skin model using six strains of MRSA representing different clonal types, namely, ST8, ST30, ST59, ST22, ST45 and ST239. All the six strains exhibited internalization in HaCaT cells. Due to cell detachment, the invasion study was limited up to two and a half hours. TUNEL assay showed no significant difference in the cell death induced by the six MRSA strains in the HaCaT cells. Our 3D-skin model provided a better insight into the interactions between the MRSA strains and the human skin during the infection establishment as we could study the infection of MRSA in our skin model up to 48 h. Immunohistochemical staining together with TUNEL assay in the 3D-skin model showed co-localization of the bacteria with the apoptotic cells demonstrating the induction of apoptosis by the bacteria and revealed the variation in bacterial transmigration among the MRSA strains. The strain representing ST59 showed maximum internalization in HaCaT cells and the maximum cell death as measured by Apoptotic index in the 3D-skin model. Our results show that 3D-skin model might be more likely to imitate the physiological response of skin to MRSA infection than 2D-HaCaT monolayer keratinocyte cultures and will enhance our understanding of the difference in pathogenesis among different MRSA strains.

Colonization of Group B Streptococcus in Pregnant Women and Their Neonates from a Sri Lankan Hospital.

We investigated the molecular epidemiology of Streptococcus agalactiae (Group B Streptococcus, GBS) from carriage in a cohort of pregnant mothers and their respective newborns in a Teaching Hospital in Sri Lanka. GBS vaginal carriage was assessed on pregnant mothers at pre-delivery (n = 250), post-delivery (n = 130), and from peri-rectal swabs of neonates (n = 159) in a prospective study. All colonizing, non-duplicate GBS isolates (n = 60) were analyzed for antimicrobial susceptibilities, capsular serotyping, and whole-genome sequencing (WGS). The percentage of GBS carriage in mothers in the pre-delivery and post-delivery cohorts were 11.2% (n = 28) and 19.2% (n = 25), respectively, and 4.4% (n = 7) in neonates. GBS isolates predominantly belonged to serotype VI (17/60, 28.3%). The isolates spanned across 12 sequence types (STs), with ST1 (24/60, 40%) being the most predominant ST. Concomitant resistance to erythromycin, tetracyclines, and gentamicin was observed in eight strains (13.3%). WGS revealed the presence of antimicrobial resistance genes including ermA (5/60), mefA (1/60), msrD (1/60), and tetLMO (2/60, 28/60, and 1/60, respectively) among 60 strains. The study provides insight into the diversity of vaccine targets of GBS since serotype VI is yet to be covered in the vaccine development program.

Prevalence and Characteristics of Streptococcus agalactiae from Freshwater Fish and Pork in Hong Kong Wet Markets.

We report the antimicrobial resistance of 191 fish and 61 pork Group B Streptococcus (GBS) procured from Hong Kong wet markets. Two-hundred-and-fifty-two GBS strains were isolated from 992 freshwater fish and 361 pig offal during 2016-2019. The strains were isolated from homogenised samples and plated on selective media, followed by identification through MALDI-TOF-MS. Molecular characterisation, an antibiotic susceptibility test, and biofilm formation were performed on the strains. The isolation rates of the fish GBS and pig GBS were 19.3% (191 strains from 992 freshwater fish) and 16.9% (61 strains from 361 pig organs), respectively. The fish GBS was predominantly serotype Ia, ST7, while pig GBS was serotype III, ST651 (45 strains). An antibiotic susceptibility test revealed that the fish GBS were mostly antibiotic-sensitive, while the pig GBS were multidrug-resistant. A biofilm formation experiment showed that over 71% of fish GBS and all pig GBS had moderate biofilm formation ability. In general, the prevalence rate of GBS in animals and the multidrug resistance phenotype presented in the strains raise concerns about its zoonotic potential and effects on public health.

Correction: Li et al. Multidrug-Resistant Streptococcus agalactiae Strains Found in Human and Fish with High Penicillin and Cefotaxime Non-Susceptibilities. Microorganisms 2020, 8, 1055.

The authors would like to make the following correction to the published paper [...].

Multidrug-Resistant Streptococcus agalactiae Strains Found in Human and Fish with High Penicillin and Cefotaxime Non-Susceptibilities.

Penicillin non-susceptible Streptococcus agalactiae (PEN-NS GBS) has been increasingly reported, with multidrug-resistant (MDR) GBS documented in Japan. Here we identified two PEN-NS GBS strains during our surveillance studies: one from a patient's wound and the other from a tilapia. The patient's GBS (H21) and fish GBS (F49) were serotyped and tested for antibiotic susceptibility. Whole-genome sequencing was performed to find the sequence type, antimicrobial resistance genes, and mutations in penicillin-binding proteins (PBPs) and fluoroquinolone (FQ) resistance genes. H21 and F49 belonged to ST651, serotype Ib, and ST7, serotype Ia, respectively. H21 showed PEN and cefotaxime minimum inhibitory concentrations (MICs) of 2.0 mg/L. F49 showed PEN MIC 0.5 mg/L. H21 was MDR with ermB, lnuB, tetS, ant6-Ia, sat4a, and aph3-III antimicrobial resistance genes observed. Alignment of PBPs showed the combination of PBP1B (A95D) and 2B mutations (V80A, S147A, S160A) in H21 and a novel mutation in F49 at N192S in PBP2B. Alignment of FQ-resistant determinants revealed mutation sites on gyrA, gyrB, and parC and E in H21. To our knowledge, this is the first report of GBS isolates with such high penicillin and cefotaxime MICs. This raises the concern of emergence of MDR and PEN-NS GBS in and beyond healthcare facilities.

A Small RNA Transforms the Multidrug Resistance of Pseudomonas aeruginosa to Drug Susceptibility.

Bacteria with multiple drug resistance (MDR) have become a global issue worldwide, and hundreds of thousands of people's lives are threatened every year. The emergence of novel MDR strains and insufficient development of new antimicrobial agents are the major reasons that limit the choice of antibiotics for the treatment of bacterial infection. Thus, preserving the clinical value of current antibiotics could be one of the effective approaches to resolve this problem. Here we identified numerous novel small RNAs that were downregulated in the MDR clinical isolates of Pseudomonas aeruginosa (P. aeru), and we demonstrated that overexpression of one of these small RNAs (sRNAs), AS1974, was able to transform the MDR clinical strain to drug hypersusceptibility. AS1974 is the master regulator to moderate the expression of several drug resistance pathways, including membrane transporters and biofilm-associated antibiotic-resistant genes, and its expression is regulated by the methylation sites located at the 5' UTR of the gene. Our findings unravel the sRNA that regulates the MDR pathways in clinical isolates of P. aeru. Moreover, transforming bacterial drug resistance to hypersusceptibility using sRNA could be the potential approach for tackling MDR bacteria in the future.

Comparative transcriptomics of multidrug-resistant Acinetobacter baumannii in response to antibiotic treatments.

Multidrug-resistant Acinetobacter baumannii, a major hospital-acquired pathogen, is a serious health threat and poses a great challenge to healthcare providers. Although there have been many genomic studies on the evolution and antibiotic resistance of this species, there have been very limited transcriptome studies on its responses to antibiotics. We conducted a comparative transcriptomic study on 12 strains with different growth rates and antibiotic resistance profiles, including 3 fast-growing pan-drug-resistant strains, under separate treatment with 3 antibiotics, namely amikacin, imipenem, and meropenem. We performed deep sequencing using a strand-specific RNA-sequencing protocol, and used de novo transcriptome assembly to analyze gene expression in the form of polycistronic transcripts. Our results indicated that genes associated with transposable elements generally showed higher levels of expression under antibiotic-treated conditions, and many of these transposon-associated genes have previously been linked to drug resistance. Using co-expressed transposon genes as markers, we further identified and experimentally validated two novel genes of which overexpression conferred significant increases in amikacin resistance. To the best of our knowledge, this study represents the first comparative transcriptomic analysis of multidrug-resistant A. baumannii under different antibiotic treatments, and revealed a new relationship between transposons and antibiotic resistance.