Brett E. Swierczewski

Background/Objectives: Phages show efficacy against multidrug-resistant Pseudomonas aeruginosa, but limited host ranges require combining them in cocktails. In this work, we characterized 25 P. aeruginosa phages, developed therapeutic cocktails active against diverse clinical isolates, and tested phage efficacy in a mouse incisional wound model. Methods/Results: These phages represent seven genera, and genomic and phenotypic analyses indicate that 24/25 are lytic and suitable for phage therapy. Phage host ranges on a diversity panel of 156 P. aeruginosa strains that included 106 sequence types varied from 8% to 54%, and together the 24 lytic phages were active against 133 strains (85%). All of the phages reduced bacterial counts in biofilms. A cocktail of five lytic phages, WRAIR_PAM1, covered 56% of the strain panel, protected 100% of mice from lethal systemic infection (vs. 20% survival in the saline-treated group), and accelerated healing of infected wounds. An improved five-phage cocktail, WRAIR_PAM2, was formulated by a rational design approach (using phages with broader host ranges, more complementing activity, relatively low resistance background, and compatibility in mixes). Conclusions: WRAIR_PAM2 covered 76% of highly diverse clinical isolates and demonstrated significant efficacy against topical and systemic P. aeruginosa infection, indicating that it is a promising therapeutic candidate.

Phages show efficacy against multidrug-resistant Pseudomonas aeruginosa, but limited host ranges require combining them in cocktails. In this work, we characterized 25 P. aeruginosa phages, developed therapeutic cocktails active against diverse clinical isolates, and tested phage efficacy in a mouse incisional wound model. These phages represent seven genera, and genomic and phenotypic analyses indicate that 24/25 are lytic and suitable for phage therapy. Phage host ranges on a diversity panel of 156 P. aeruginosa strains that included 106 sequence types varied from 8% to 54%, and together the 24 lytic phages were active against 133 strains (85%). All of the phages reduced bacterial counts in biofilms. A cocktail of five lytic phages, WRAIR_PAM1, covered 56% of the strain panel, protected 100% of mice from lethal systemic infection (vs. 20% survival in the saline-treated group) and accelerated healing of infected wounds. An improved 5-phage cocktail, WRAIR_PAM2, was formulated by a rational design approach (using phages with broader host ranges, more complementing activity, relatively low resistance background, and compatibility in mixes). WRAIR_PAM2 covered 76% of highly diverse clinical isolates and demonstrated significant efficacy against topical and systemic P. aeruginosa infection, indicating that it is a promising therapeutic candidate.

The emergence of colistin resistance poses a significant threat to its efficacy as a last-line treatment against multidrug-resistant Gram-negative bacterial infections. In this study, 178 multi-drug resistant (MDR) Escherichia coli isolates collected from clinical samples at Queen Sirikit Naval Hospital, Chonburi, Thailand, were evaluated for colistin resistance. Of these, six were identified as mcr gene carriers, mediating colistin resistance. Specifically, mcr-1 was detected in three E. coli isolates, mcr-3 was detected in one E. coli isolate, and mcr-1 and mcr-3 were detected in two E. coli isolates, designated AMR-0220 and AMR-0361. Whole-genome sequencing and bioinformatics analysis revealed that AMR-0220 and AMR-0361 belonged to ST410 and ST617 lineages, respectively. Both isolates carried multiple plasmids, with mcr-1.1 located on an IncX4-type plasmid that is closely related to previously reported mcr-1.1-carrying IncX4 plasmids. In contrast, mcr-3.5 was identified on distinct plasmid backbones: an IncFIB-type plasmid in AMR-0220 and an IncFII-type plasmid in AMR-0361. Overall, our findings demonstrate that the mcr genes found in E. coli isolates in this region are located on different mobile genetic elements, indicating the potential for a widespread dissemination of colistin resistance among Gram-negative bacteria throughout Thailand’s healthcare system.

Staphylococcus aureus (S. aureus), especially methicillin-resistant S. aureus (MRSA), remains a major public health concern. This study reports the antimicrobial resistance profiles and molecular characteristics of 31 S. aureus isolated during 2017–2018 from inpatient and outpatient clinical specimens from Queen Sirikit Naval Hospital (QSH) in Chonburi province, Thailand. All isolates were tested for antimicrobial susceptibility. Staphylococcal cassette chromosome mec (SCCmec) typing, Panton–Valentine leukocidin (pvl) toxin, pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and staphylococcal protein A (spa) typing were performed. Twenty-seven isolates were confirmed to be MRSA and exhibited resistance to up to seven antibiotics classes. The main MLST type was SCCmec type II (51.9%) and ST764 (55.6%). Five spa types were identified with t045 (55.6%) as the major type. All 31 S. aureus isolates were grouped into seven types using PFGE with the SCCmecII-ST764-t045 clone being the most prevalent. Overall, our findings reveal that the S. aureus isolates in this study differ from previous reports in Thailand, indicating a potential shift in local strains, highlighting the need for ongoing molecular surveillance of multidrug resistance patterns of MRSA in Southeast Asia.

Shigellosis is a leading global cause of diarrheal disease and travelers’ diarrhea now being complicated by the dissemination of antibiotic resistance, necessitating the development of alternative antibacterials such as therapeutic bacteriophages (phages). Phages with lytic activity against Shigella strains were isolated from sewage. The genomes of 32 phages were sequenced, and based on genomic comparisons belong to seven taxonomic genera: Teetrevirus, Teseptimavirus, Kayfunavirus, Tequatrovirus, Mooglevirus, Mosigvirus and Hanrivervirus. Phage host ranges were determined with a diverse panel of 95 clinical isolates of Shigella from Southeast Asia and other geographic regions, representing different species and serotypes. Three-phage mixtures were designed, with one possessing lytic activity against 89% of the strain panel. This cocktail exhibited lytic activity against 100% of S. sonnei isolates, 97.2% of S. flexneri (multiple serotypes) and 100% of S. dysenteriae serotypes 1 and 2. Another 3-phage cocktail composed of two myophages and one podophage showed both a broad host range and the ability to completely sterilize liquid culture of a model virulent strain S. flexneri 2457T. In a Galleria mellonella model of lethal infection with S. flexneri 2457T, this 3-phage cocktail provided a significant increase in survival.

Antibiotic resistance, when it comes to bacterial infections, is not a problem that is going to disappear anytime soon. With the lack of larger investment in novel antibiotic research and the ever-growing increase of resistant isolates amongst the ESKAPEE pathogens (Enterobacter cloacae, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterococcus sp., and Escherichia coli), it is inevitable that more and more infections caused by extensively drug-resistant (XDR) and pandrug-resistant (PDR) strains will arise. One strategy to counteract the growing threat is to use antibiotic adjuvants, a drug class that on its own lacks significant antibiotic activity, but when mixed with another antibiotic, can potentiate increased killing of bacteria. Antibiotic adjuvants have various mechanisms of action, but polymyxins and polymyxin-like molecules can disrupt the Gram-negative outer membrane and allow other drugs better penetration into the bacterial periplasm and cytoplasm. Previously, we showed that SPR741 had this adjuvant effect with regard to rifampin; however, rifampin is often not used clinically because of easily acquired resistance. To find additional, appropriate clinical partners for SPR741 with respect to pulmonary and wound infections, we investigated tetracyclines and found a previously undocumented synergy with minocycline in vitro and in vivo in murine models of infection.

Schistosoma mekongi is one of five major causative agents of human schistosomiasis and is endemic to communities along the Mekong River in southern Lao People’s Democratic Republic (Laos) and northern Cambodia. Sporadic cases of schistosomiasis have been reported in travelers and immigrants who have visited endemic areas. Schistosoma mekongi biology and molecular biology is poorly understood, and few S. mekongi gene and transcript sequences are available in public databases. Transcriptome sequencing (RNA-Seq) of male and female S. mekongi adult worms (a total of three biological replicates for each sex) were analyzed and the results demonstrated that approximately 304.9 and 363.3 million high-quality clean reads with quality Q30 (> 90%) were obtained from male and female adult worms, respectively. A total of 119,604 contigs were assembled with an average length of 1273 nt and an N50 of 2017 nt. From the contigs, 20,798 annotated protein sequences and 48,256 annotated transcript sequences were obtained using BLASTP and BLASTX searches against the UniProt Trematoda database. A total of 4658 and 3509 transcripts were predominantly expressed in male and female worms, respectively. Male-biased transcripts were mostly involved in structural organization while female-biased transcripts were typically involved in cell differentiation and egg production. Interestingly, pathway enrichment analysis suggested that genes involved in the phosphatidylinositol signaling pathway may play important roles in the cellular processes and reproductive systems of S. mekongi worms. We present comparative transcriptomic analyses of male and female S. mekongi adult worms, which provide a global view of the S. mekongi transcriptome as well as insights into differentially-expressed genes associated with each sex. This work provides valuable information and sequence resources for future studies of gene function and for ongoing whole genome sequencing efforts in S. mekongi.

Leptospirosis, a global neglected zoonotic disease, is an important public health problem in Thailand. Nonspecific symptoms, lack of laboratory confirmation, and underreporting contribute to its neglected disease status. To better understand the distribution of leptospirosis exposure in Thailand, a retrospective leptospirosis seroprevalence study was conducted on repository serum specimens obtained from young Thai men entering the Royal Thai Army during 2007–2008. The overall nationwide leptospirosis IgG seroprevalence among these young Thai men was 28% (95% confidence interval = 26–30%) and the range by province was 10–52% confirming leptospirosis as an endemic disease throughout Thailand. Seroprevalence was highest in individuals with the lowest education from rural areas, and higher seroprevalence was found in the north and south regions contrary to current morbidity reports. Improvement in reporting and surveillance as well as better access to leptospirosis diagnostics will increase leptospirosis awareness and detection and enable more effective public health interventions.

Children with acute and chronic malnutrition are at increased risk of morbidity and mortality following a diarrheal episode. To compare diarrheal disease severity and pathogen prevalence among children with and without acute and chronic malnutrition, we conducted a cross-sectional study of human immunodeficiency virus-uninfected Kenyan children aged 6–59 months, who presented with acute diarrhea. Children underwent clinical and anthropometric assessments and provided stool for bacterial and protozoal pathogen detection. Clinical and microbiological features were compared using log binomial regression among children with and without wasting (mid-upper arm circumference ≤ 125 mm) or stunting (height-for-age z score ≤ −2). Among 1,363 children, 7.0% were wasted and 16.9% were stunted. After adjustment for potential confounders, children with wasting were more likely than nonwasted children to present with at least one Integrated Management of Childhood Illness danger sign (adjusted prevalence ratio [aPR]: 1.3, 95% confidence interval [CI]: 1.0 to 1.5, P = 0.05), severe dehydration (aPR: 2.4, 95% CI: 1.5 to 3.8, P < 0.01), and enteroaggregative Escherichia coli recovered from their stool (aPR: 1.8, 1.1–2.8, P = 0.02). There were no differences in the prevalence of other pathogens by wasting status after confounder adjustment. Stunting was not associated with clinical severity or the presence of specific pathogens. Wasted children with diarrhea presented with more severe disease than children without malnutrition which may be explained by a delay in care-seeking or diminished immune response to infection. Combating social determinants and host risk factors associated with severe disease, rather than specific pathogens, may reduce the disparities in poor diarrhea-associated outcomes experienced by malnourished children.

Campylobacter concisus and C. ureolyticus have emerged in recent years as being associated with acute and prolonged gastroenteritis and implicated in the development of inflammatory bowel diseases. However, there are limited data on the prevalence of these microorganisms in Southeast Asia. In this study, 214 pathogen-negative stool samples after laboratory examination for common enteric pathogens to include C. jejuni and C. coli by culture from two case–control traveler’s diarrhea (TD) studies conducted in Thailand (cases = 26; controls = 30) and Nepal (cases = 83; controls = 75) respectively were assayed by PCR for the detection of Campylobacter 16S rRNA and two specific heat shock protein genes specific for C. concisus (cpn60) and C. ureolyticus (Hsp60) respectively. Campylobacter 16S rRNA was detected in 28.5% (61/214) of the pathogen-negative TD stool samples (CIWEC Travel Medicine Clinic, Kathmandu, Nepal: cases = 36, control = 14; Bamrungrad International Hospital, Bangkok, Thailand: cases = 9, controls = 2). C. consisus was identified significantly more often in TD cases in Nepal (28.9%; 24/83) as compared to controls (4%; 3/75) (OR = 9.76; 95% CI 2.80–34.02; P = 0.0003) while C. consisus was detected in only two cases (2/26; 7.7%) and none of the controls stool samples from Thailand. C. ureolyticus was detected in four cases (4.8%; 4/83) and four controls (5.3%; 4/75) and in one case (3.8%; 1/26) and one control (3.1%; 1/30) from Nepal and Thailand respectively. C. jejuni and C. coli were isolated in 18.3 and 3.4% of the cases and in 4.0 and 1.4% of the controls in stool samples from both Thailand and Nepal respectively while C. concisus nor C. ureolyticus were not tested for in these samples. These findings suggest that C. concisus potentially is a pathogen associated with TD in Nepal. To our knowledge, this is the first report of C. concisus and C. ureolyticus detected from traveler’s diarrhea cases from travelers to Nepal and Thailand.