Antimicrobial Peptide Mastoparan-AF Kills Multi-Antibiotic Resistant Escherichia coli O157:H7 via Multiple Membrane Disruption Patterns and Likely by Adopting 3-11 Amphipathic Helices to Favor Membrane Interaction.

We investigated the antimicrobial activity and membrane disruption modes of the antimicrobial peptide mastoparan-AF against hemolytic Escherichia coli O157:H7. Based on the physicochemical properties, mastoparan-AF may potentially adopt a 3-11 amphipathic helix-type structure, with five to seven nonpolar or hydrophobic amino acid residues forming the hydrophobic face. E. coli O157:H7 and two diarrheagenic E. coli veterinary clinical isolates, which are highly resistant to multiple antibiotics, are sensitive to mastoparan-AF, with minimum inhibitory and bactericidal concentrations (MIC and MBC) ranging from 16 to 32 µg mL-1 for E. coli O157:H7 and four to eight µg mL-1 for the latter two isolates. Mastoparan-AF treatment, which correlates proportionally with membrane permeabilization of the bacteria, may lead to abnormal dents, large perforations or full opening at apical ends (hollow tubes), vesicle budding, and membrane corrugation and invagination forming irregular pits or pores on E. coli O157:H7 surface. In addition, mRNAs of prepromastoparan-AF and prepromastoparan-B share a 5'-poly(A) leader sequence at the 5'-UTR known for the advantage in cap-independent translation. This is the first report about the 3-11 amphipathic helix structure of mastoparans to facilitate membrane interaction. Mastoparan-AF could potentially be employed to combat multiple antibiotic-resistant hemolytic E. coli O157:H7 and other pathogenic E. coli.

Genomic investigation of emerging zoonotic pathogen Shewanella xiamenensis.

Objective: Shewanella xiamenensis is an emerging zoonotic pathogen commonly found in aquatic ecosystem. Clustered regularly interspaced short palindromic repeats (CRISPR) and (CRISPR)-associated gene systems act as adaptive immune system of prokaryotes. Recently, growing evidence suggested their role in bacterial virulence and resistance. Despite its medical importance, little is known about the genomic characteristics of S. xiamenensis. Materials and Methods: Strain ZYW6 was isolated from Epinephelus awoara. We sequenced the 16S rRNA gene and blast against the GenBank bacterial database. Antibiotic susceptibility tests and interpretation were performed by automatic VITEK 2 system. We extracted the genomic DNA with QIAGEN Genomic-tip 100/G kit and QIAGEN Genomic DNA Buffer Set. Whole-genome shotgun sequencing was performed using the Illumina MiSeq sequencer. To identify the CRISPR-Cas System in the genome of S. xiamenensis ZYW6, the Integrated Microbial Genomes and Microbiomes and CRISPRFinder were used. Results: We characterized the genome of a S. xiamenensis strain. The genome is 4,765,190 bp in length and encodes 4262 open-reading frames. Type I CRISPR-Cas system and serine biosynthesis genes were identified. Conclusion: Our results demonstrate the genetic structure of CRISPR-Cas system, l-serine synthesis, and oxacillinase in S. xiamenensis. The report of antibiotics resistance genes in the study might indicate a possible reservoir of antimicrobial drug resistance determinants in food animal, resulting in potential infection source. The findings provide insights into the structure and composition of CRISPR-Cas system in S. xiamenensis and foundation for future biological validation.

Most commensally bacterial strains in human milk of healthy mothers display multiple antibiotic resistance.

Recent reports have shown that food-borne or commensal bacteria can function as reservoirs of antibiotic resistance. However, the antibiotic susceptibility of bacterial isolates of most milk samples or the total bacterial counts (TBC) in human milk from healthy donors, are not fully understood in Taiwan. Thus, five healthy mothers were randomly recruited each month, and totally 30 mothers without any symptoms of infection were recruited over 6 months. Milk samples were then harvested and analyzed immediately after collection. The antibiotic susceptibility was analyzed in bacteria isolated from milk samples using nine clinically relevant antibiotics, such as oxacillin, ampicillin, cephalothin, amoxicillin, ciprofloxacin, erythromycin, clindamycin, gentamicin, and oxytetracycline. The Staphylococcus strains (48 isolates) found in milk resisted to 48.6 ± 20.1% selected antibiotics. Streptococcus-related isolates (8 isolates) exhibited resistance to 41.7 ± 26.4% selected antibiotics. Acinetobacter isolates (5 isolates) were resistant to 66.7 ± 13.6% antibiotics, and Enterococcus isolates (5 isolates) were resistant to 73.3 ± 6.1% tested antibiotics. Rothia-related isolates (4 isolates) were resisted to 58.2 ± 31.9% of tested antibiotics. In contrast, Corynebacterium isolates (5 isolates) were sensitive to 66%-100% of selected antibiotics. Furthermore, the TBC ranged from 40 to 710,000 CFU/ml, implying a wide spectrum of bacteria in milk from healthy mothers. Despite this, all milk donors were healthy during sampling, and they did not show any symptoms related to mastitis or subclinical mastitis. According to the previously described TBC criteria for the use of donated human milk, only 73% of the current milk samples could be accepted for the milk bank. In conclusion, the majority of the isolated bacterial strains from current human milk samples are multiresistant strains. In milk samples for preterm infants or milk banks, higher TBC levels or potentially antibiotic-resistant bacteria in some milk samples have supported people using approaches to disinfect human milk partially.

The Pathogenicity of Shewanella algae and Ability to Tolerate a Wide Range of Temperatures and Salinities.

Shewanella algae is a rod-shaped Gram-negative marine bacterium frequently found in nonhuman sources such as aquatic ecosystems and has been shown to be the pathogenic agent in various clinical cases due to the ingestion of raw seafood. The results of this study showed that S. algae was present in approximately one in four samples, including water and shellfish samples. Positive reactions (API systems) in S. algae strains were seen for gelatinase (gelatin); however, negative reactions were found for indole production (tryptophan). S. algae is adapted to a wide range of temperatures (4°C, 25°C, 37°C, and 42°C) and salinity. Temperature is a key parameter in the pathogenicity of S. algae as it appears to induce hemolysis at 25°C and 37°C. S. algae exhibits pathogenic characteristics at widely varying temperatures, which suggests that it may have the ability to adapt to climate change.

Genome characterization of bile-isolated Shewanella algae ACCC.

BACKGROUND: Shewanella algae has been recognized as an emerging human pathogen. However, not much is known about the mechanism of its pathogenesis and its adaptation to a special niche such as the hepatobiliary tract. RESULTS: In this study, we isolated the S. algae ACCC strain from human bile and performed whole genome sequencing. S. algae ACCC consists of a circular 4,743,354-bp chromosome with a GC content of 53.08%, within 4080 protein coding sequences. The genome of strain ACCC contains a number of candidate genes which have been reported to be associated with bile adaption, including htpB, exbBD, wecA, galU, adeFGH and phoPQ regulon. CONCLUSIONS: Our results highlight the association of S. algae with a rare disease profile. Further studies are needed to shed light on the evolution of pathogenesis and the niche adaptation of S. algae.

Characteristics and Phylogeny of Shewanella haliotis Isolated from Cultivated Shellfish in Taiwan.

Shewanella haliotis is an emerging human pathogen. Many infectious cases were linked to shellfish ingestion or aquatic exposure. Therefore, it is important to study the phylogeny and distribution of S. haliotis in shellfish aquaculture. We investigated the distribution of S. haliotis in cultivated shellfish farming in Taiwan in which S. haliotis was found in the shellfish from all sampling sites. S. haliotis was identified in cultivated shellfish by 16S rRNA gene sequencing, such as abalone (Haliotis diversicolor), clam (Meretrix lusoria), and oyster (Crassostrea gigas). This study highlighted the contamination of S. haliotis in cultivated shellfish and importance of further study regarding the biodiversity and pathogenesis of S. haliotis.

Volatile Oils of Nepeta tenuifolia (Jing Jie) as an Alternative Medicine against Multidrug-Resistant Pathogenic Microbes.

Essential oils from the dried spikes of Nepeta tenuifolia (Benth) are obtained by steam distillation. Pulegone was identified as the main component in the spikes of N. tenuifolia through analysis, with greater than 85% purity obtained in this study. The essential oils are extremely active against all Gram-positive and some Gram-negative reference bacteria, particularly Salmonella enterica, Citrobacter freundii, and Escherichia coli. The minimum inhibitory concentration was found to be between 0.08 and 0.78% (against S. enterica), 0.39 and 0.78% (against C. freundii), and 0.097 and 0.39% (against E. coli), whereas the minimum bactericidal concentration varied in range from 0.097% to 1.04%. In general, the essential oils show a strong inhibitory action against all tested reference strains and clinical isolates. However, the antibacterial activity of EOs against both Pseudomonas aeruginosa reference strains and clinical isolates was relatively lower than other Gram-negative pathogens. The essential oils of N. tenuifolia also displayed bactericidal activities (MBC/MIC < 4) in this study. These findings reflect the bactericidal activity of the essential oils against a wide range of multidrug-resistant clinical pathogens in an in vitro study. In addition, we propose the fragmentation pathways of pulegone and its derivatives by LC-ESI-MS/MS in this study.

Colistin Resistance of Pseudomonas aeruginosa Isolated from Snakes in Taiwan.

This study included fifty-eight isolates of P. aeruginosa from the oral cavity of snakes that were recruited from clinical cases, captive and wild snakes. The minimum inhibitory concentrations (MICs) for the determination of susceptibility were identified by the broth microdilution method. Polymerase chain reaction (PCR) was employed to detect ß-lactamases genes. With regard to antipseudomonal antibiotics, the lowest nonsusceptible rates were in aztreonam (15%), piperacillin/tazobactam (12%), and amikacin (9%). The nonsusceptible rates were high in gentamicin (33%) and colistin (55%). Meanwhile, blaTEM presented in 100% of isolates where blaAmpC, blaOXA-1, and blaOXA-10 came at 94.8%, 89.7%, and 27.6%, respectively. Emergence of multidrug resistant (MDR) strains and colistin-resistant strains highlights the potential breach of public health as P. aeruginosa could be transmitted through either direct contact or indirect dissemination through the environment. This study reports that the highly resistant P. aeruginosa from snakes' oral cavity were discovered for the very first time in Taiwan.

Antibiotic Susceptibility of Commensal Bacteria from Human Milk.

Recent studies have focused on foodborne or commensal bacteria as vehicles of antibiotic resistance. However, the antibiotic resistance of milk bacteria from healthy donors is still vague in Taiwan. For this purpose, human milk samples were obtained from randomly recruited 19 healthy women between 3 and 360 days post-partum. Antibiotic susceptibility profile of bacteria from milk samples was determined. About 20 bacterial species were isolated from milk samples including Staphylococcus (6 species), Streptococcus (4 species), Enterococcus (2 species), Lactobacillus (1 species), and bacteria belonging to other genera (7 species). Some opportunistic or potentially pathogenic bacteria including Kluyvera ascorbata, Klebsiella oxytoca, Klebsiella pneumoniae, Acinetobacter baumannii, Actinomyces bovis, and Staphylococcus aureus were also isolated. Intriguingly, Staphylococcus isolates (22 strains) were resistant to 2­8 of 8 antibiotics, while Streptococcus isolates (3 strains) were resistant to 3­7 of 9 antibiotics, and members of the genus Enterococcus (5 strains) were resistant to 3­8 of 9 antibiotics. Notably, Staphylococcus lugdunensis, S. aureus, Streptococcus parasanguinis, Streptococcus pneumonia, and Enterococcus faecalis were resistant to vancomycin, which is considered as the last-resort antibiotic. Therefore, this study shows that most bacterial strains in human milk demonstrate mild to strong antibiotic resistance. Whether commensal bacteria in milk could serve as vehicles of antibiotic resistance should be further investigated.