Complete genome sequence of the broad host range Acinetobacter baumannii phage EAb13.

We describe the genome of a lytic phage EAb13 isolated from sewage, with broad activity against multidrug-resistant Acinetobacter baumannii. EAb13 is an unclassified siphovirus. Its genome consists of 82,411 bp, with 40.15% GC content, 126 protein-coding sequences, 1 tRNA, and 2,177 bp-long direct terminal repeats.

Genome Sequence of Staphylococcus aureus Phage ESa2.

We describe the genome of a lytic phage, ESa2, isolated from environmental water and specific for Staphylococcus aureus. ESa2 belongs to the family Herelleviridae and genus Kayvirus. Its genome consists of 141,828 bp, with 30.25% GC content, 253 predicted protein-coding sequences, 3 tRNAs, and 10,130-bp-long terminal repeats.

Molecular characterization of multidrug-resistant ESKAPEE pathogens from clinical samples in Chonburi, Thailand (2017-2018).

BACKGROUND: ESKAPEE pathogens Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp. and Escherichia coli are multi-drug resistant (MDR) bacteria that present increasing treatment challenges for healthcare institutions and public health worldwide. METHODS: 431 MDR ESKAPEE pathogens were collected from Queen Sirikit Naval Hospital, Chonburi, Thailand between 2017 and 2018. Species identification and antimicrobial resistance (AMR) phenotype were determined following CLSI and EUCAST guidelines on the BD Phoenix System. Molecular identification of antibiotic resistant genes was performed by polymerase chain reaction (PCR), real-time PCR assays, and whole genome sequencing (WGS). RESULTS: Of the 431 MDR isolates collected, 1.2% were E. faecium, 5.8% were S. aureus, 23.7% were K. pneumoniae, 22.5% were A. baumannii, 4.6% were P. aeruginosa, 0.9% were Enterobacter spp., and 41.3% were E. coli. Of the 401 Gram-negative MDR isolates, 51% were carbapenem resistant, 45% were ESBL producers only, 2% were colistin resistance and ESBLs producers (2%), and 2% were non-ESBLs producers. The most prevalent carbapenemase genes were blaOXA-23 (23%), which was only identified in A. baumannii, followed by blaNDM (17%), and blaOXA-48-like (13%). Beta-lactamase genes detected included blaTEM, blaSHV, blaOXA, blaCTX-M, blaDHA, blaCMY, blaPER and blaVEB. Seven E. coli and K. pneumoniae isolates showed resistance to colistin and carried mcr-1 or mcr-3, with 2 E. coli strains carrying both genes. Among 30 Gram-positive MDR ESKAPEE, all VRE isolates carried the vanA gene (100%) and 84% S. aureus isolates carried the mecA gene. CONCLUSIONS: This report highlights the prevalence of AMR among clinical ESKAPEE pathogens in eastern Thailand. E. coli was the most common MDR pathogen collected, followed by K. pneumoniae, and A. baumannii. Carbapenem-resistant Enterobacteriaceae (CRE) and extended spectrum beta-lactamases (ESBLs) producers were the most common resistance profiles. The co-occurrence of mcr-1 and mcr-3 in 2 E. coli strains, which did not affect the level of colistin resistance, is also reported. The participation of global stakeholders and surveillance of MDR remain essential for the control and management of MDR ESKAPEE pathogens.

Identification and Characterization of vB_PreP_EPr2, a Lytic Bacteriophage of Pan-Drug Resistant Providencia rettgeri.

Providencia rettgeri is an emerging opportunistic Gram-negative pathogen with reports of increasing antibiotic resistance. Pan-drug resistant (PDR) P. rettgeri infections are a growing concern, demonstrating a need for the development of alternative treatment options which is fueling a renewed interest in bacteriophage (phage) therapy. Here, we identify and characterize phage vB_PreP_EPr2 (EPr2) with lytic activity against PDR P. rettgeri MRSN 845308, a clinical isolate that carries multiple antibiotic resistance genes. EPr2 was isolated from an environmental water sample and belongs to the family Autographiviridae, subfamily Studiervirinae and genus Kayfunavirus, with a genome size of 41,261 base pairs. Additional phenotypic characterization showed an optimal MOI of 1 and a burst size of 12.3 ± 3.4 PFU per bacterium. EPr2 was determined to have a narrow host range against a panel of clinical P. rettgeri strains. Despite this fact, EPr2 is a promising lytic phage with potential for use as an alternative therapeutic for treatment of PDR P. rettgeri infections.

First Report: Colistin Resistance Gene mcr-3.1 in Salmonella enterica Serotype Choleraesuis Isolated from Human Blood Sample from Thailand.

This study describes the first finding of Salmonella enterica serotype Choleraesuis (Salmonella Choleraesuis) isolate harboring mobile colistin resistance (mcr)-3.1 obtained from human blood sample. The clinical relevant blood sample was collected during October 2018. The phenotypic identification and antimicrobial susceptibility testing (AST) were studied by using automate microbiology platform (Phoenix M50, BD), and in-depth characterization by whole genome sequencing. The phenotypic identification was reported Salmonella Choleraesuis. AST result demonstrated that this isolate had high minimum inhibitory concentrations (MICs) against colistin, fluoroquinolone, and cephalosporin III and IV, which are first-line antibiotic treatment choices for Gram-negative bacterial pathogen infections. This Salmonella Choleraesuis is harboring mcr-3.1 and presented a diversity carbapenemase including blaTEM and blactx-m-55. Regarding the multilocus sequence typing result, this Salmonella presented ST139 that related to the Choleraesuis variant sensu stricto. Swine is not the host specific for the Salmonella Choleraesuis since it also causes enteric and other diseases in human. Hence, the presence of the mobile plasmid colistin mcr-3.1 resistant gene in human sample is resulting to the public health concerns due to the fact that it is enable to transmit to other hosts and distribute into an environment.

Characterization of KPC-82, a KPC-2 Variant Conferring Resistance to Ceftazidime-Avibactam in a Carbapenem-Nonsusceptible Clinical Isolate of Citrobacter koseri.

KPC-82 is a KPC-2 variant identified in a carbapenem-nonsusceptible Citrobacter koseri that confers high-level resistance to ceftazidime-avibactam. Genomic analysis revealed that blaKPC-82 is carried by a chromosomally integrated Tn4401 transposon (disrupting porin gene phoE) and evolved by a 6-nucleotide tandem repeat duplication causing a two-amino-acid insertion (Ser-Asp) within the Ala267-Ser275 loop. Similar to related KPC variants, KPC-82 showed decreased carbapenemase activity when expressed in a heterologous background and remained susceptible to carbapenem/ß-lactamase inhibitor combinations.

First report of the mcr-1 colistin resistance gene identified in two Escherichia coli isolates from clinical samples, Philippines, 2018.

OBJECTIVES: The first report of a plasmid-borne colistin resistance gene (mcr-1) detected in an Escherichia coli isolate from China heralded the emergence of pandrug-resistant bacteria. Since then, the mcr-1 gene has been detected worldwide, but to date it has not been reported in the Philippines. METHODS: In this study, 123 antimicrobial-resistant isolates collected from January-June 2018 from patients admitted to a tertiary hospital in Manila, Philippines, were characterised. Biochemical identification and antimicrobial susceptibility testing were performed using a BD Phoenix™ M50 system with NMIC/ID-95 panel. Conventional PCR was performed to detect the genes mcr-1 to mcr-5, and short- and long-read whole-genome sequencing was performed. RESULTS: Two mcr-1-positive E. coli clinical isolates from separate patients harboured mcr-1 on an IncI2 plasmid. One isolate was shown to carry 12 antimicrobial resistance genes (ARGs) in addition to mcr-1, including the extended-spectrum ß-lactamase blaCTX-M-55, whilst the other E. coli isolate carried 6 ARGs in addition to mcr-1. CONCLUSION: Both patients had no prior colistin treatment recorded in their medical history and no travel history outside of the country within the past 6 months from the date of hospital admission, indicating local transmission and acquisition of the colistin-resistant strain from either community or hospital settings within the Philippines. This report should serve as a signal to local public-health officials of the need to intensify surveillance efforts and to increase vigilance and implementation of antimicrobial stewardship programmes to contain and slow the spread of antimicrobial resistance.

Comparison of Carbapenem-Resistant Microbial Pathogens in Combat and Non-combat Wounds of Military and Civilian Patients Seen at a Tertiary Military Hospital, Philippines (2013-2017).

INTRODUCTION: Bacterial wound infections are a danger to both military and civilian populations. The nature of injury and infection associated with combat related wounds are important in guiding antibiotic prophylaxis and empiric treatment guidelines. MATERIALS AND METHODS: The isolates were screened for drug-resistance by the MicroScan Walkaway Plus System using either the Negative Breakpoint Combo Panel (NBCP) 30 or 34 or Positive Breakpoint Combo Panel (PBPC) 20 or 23. Isolates with a minimum inhibitory concentration (MIC) of ≥8 µg/mL to imipenem and/or meropenem were tested for both carbapenemase production using the CarbaNP test and real-time PCR to determine molecular resistance mechanisms. Plasmid conjugation analysis was performed to define potential for horizontal gene transfer. RESULTS: We characterized 634 bacterial wound isolates collected from September 2013 to December 2017 from patients seen at a Philippine military tertiary hospital presenting with combat or non-combat injuries [354 (military) and 280 (civilians)]. Staphylococcus aureus was the most predominant bacterial species isolated from wounds in both populations (104/634, 16%). A variety of Gram-negative bacterial species comprised 442/634 (70%) of the isolates identified, with the most prevalent shown to be Pseudomonas aeruginosa, Enterobacter cloacae, Klebsiella pneumoniae, Escherichia coli, and Acinetobacter sp. Carbapenemase production was detected in 34/442 (8%) Gram-negative isolates. Testing for molecular resistance mechanisms showed 32/34 (17 military, 15 civilian) wound isolates were blaNDM positive and 2 were blaVIM positive, with the two blaVIM isolates found in the civilian population. Plasmid conjugation of 14 blaNDM and 2 blaVIM positive wound isolates representatives showed 2/16 (13%) produced E. coli J53 transconjugants (E. coli from a civilian; E. cloacae from a military). CONCLUSION: We describe in this study the wound bacterial and antibiotic resistance profile in the military (combat vs non-combat associated) and civilian population. We observed that, with the exception of Acinetobacter sp., resistance of prevalent Gram-negative bacterial species to imipenem or meropenem were not significantly different between the military and civilian populations. We also presented data on the prevalent bacterial species isolated from both combat and non-combat wounds in a military tertiary care hospital setting as well as the carbapenemase-encoding gene primarily responsible for carbapenem resistance as well as evidence of horizontal transfer via mobile genetic elements. Clinicians may use this information to guide empiric antibiotic coverage for the predominant organisms if wound culture results are not readily available.A prospective, longitudinal evaluation of the wound bacterial profile documenting the changing bacterial flora using higher resolution molecular strategies can provide a more comprehensive understanding of the diversity, composition, and abundance of bacterial composition of the wound microbial community from the time of injury, during the course of evacuation from the field to higher level of care facilities, and up to wound resolution.

Mechanistic insights and therapeutic opportunities of antimicrobial chemokines.

Chemokines are a family of small proteins best known for their ability to orchestrate immune cell trafficking and recruitment to sites of infection. Their role in promoting host defense is multiplied by a number of additional receptor-dependent biological activities, and most, but not all, chemokines have been found to mediate direct antimicrobial effects against a broad range of microorganisms. The molecular mechanism(s) by which antimicrobial chemokines kill bacteria remains unknown; however, recent observations have expanded our fundamental understanding of chemokine-mediated bactericidal activity to reveal increasingly diverse and complex actions. In the current review, we present and consider mechanistic insights of chemokine-mediated antimicrobial activity against bacteria. We also discuss how contemporary advances are reshaping traditional paradigms and opening up new and innovative avenues of research with translational implications. Towards this end, we highlight a developing framework for leveraging chemokine-mediated bactericidal and immunomodulatory effects to advance pioneering therapeutic approaches for treating bacterial infections, including those caused by multidrug-resistant pathogens.

Extended-spectrum ß-lactamase prevalence and virulence factor characterization of enterotoxigenic Escherichia coli responsible for acute diarrhea in Nepal from 2001 to 2016.

Background: Multidrug-resistant (MDR) Gram-negative bacterial species are an increasingly dangerous public health threat, and are now endemic in many areas of South Asia. However, there are a lack of comprehensive data from many countries in this region determining historic and current MDR prevalence. Enterotoxigenic Escherichia coli (ETEC) is a leading cause of both acute infant diarrhea and traveler's diarrhea in Nepal. The MDR prevalence and associated resistance mechanisms of ETEC isolates responsible for enteric infections in Nepal are largely unknown. Methods: A total of 265 ETEC isolates were obtained from acute diarrheal samples (263/265) or patient control samples (2/265) at traveler's clinics or regional hospitals in Nepal from 2001 to 2016. Isolates were screened for antibiotic resistance, to include extended spectrum beta-lactamase (ESBL) production, via the Microscan Automated Microbiology System. ETEC virulence factors, specifically enterotoxins and colonization factors (CFs), were detected using multiplex PCR, and prevalence in the total isolate population was compared to ESBL-positive isolates. ESBL-positive isolates were assessed using multiplex PCR for genetic markers potentially responsible for observed resistance. Results: A total of 118/265 (44.5%) ETEC isolates demonstrated resistance to ≥2 antibiotics. ESBL-positive phenotypes were detected in 40/265 isolates, with isolates from 2008, 2013, 2014, and 2016 demonstrating ESBL prevalence rates of 1.5, 34.5, 31.2, and 35.0% respectively. No difference was observed in overall enterotoxin characterization between the total ETEC and ESBL-positive populations. The CFs CS2 (13.6%), CS3 (25.3%), CS6 (30.2%), and CS21 (62.6%) were the most prevalent in the total ETEC population. The ESBL-positive ETEC isolates exhibited a higher association trend with the CFs CS2 (37.5%), CS3 (35%), CS6 (42.5%), and CS21 (67.5%). The primary ESBL gene identified was blaCTX-M-15 (80%), followed by blaSHV-12 (20%) and blaCTX-M-14 (2.5%). The beta-lactamase genes blaTEM-1 (40%) and blaCMY-2 (2.5%) were also identified. It was determined that 42.5% of the ESBL-positive isolates carried multiple resistance genes. Conclusion: Over 30% of ETEC isolates collected post-2013 and evaluated in this study demonstrated ESBL resistance. Persistent surveillance and characterization of enteric ETEC isolates are vital for tracking the community presence of MDR bacterial species in order to recommend effective treatment strategies and help mitigate the spread of resistant pathogens.

Genomic Characterization of Nonclonal mcr-1-Positive Multidrug-Resistant Klebsiella pneumoniae from Clinical Samples in Thailand.

Multidrug-resistant Klebsiella pneumoniae strains are one of the most prevalent causes of nosocomial infections and pose an increasingly dangerous public health threat. The lack of remaining treatment options has resulted in the utilization of older drug classes, including colistin. As a drug of last resort, the discovery of plasmid-mediated colistin resistance by mcr-1 denotes the potential development of pandrug-resistant bacterial pathogens. To address the emergence of the mcr-1 gene, 118 gram-negative Enterobacteriaceae isolated from clinical samples collected at Queen Sirikit Naval Hospital in Chonburi, Thailand were screened for colistin resistance using automated antimicrobial susceptibility testing and conventional PCR screening. Two K. pneumoniae strains, QS17-0029 and QS17-0161, were positive for mcr-1, and both isolates were sequenced to closure using short- and long-read whole-genome sequencing. QS17-0029 carried 16 antibiotic resistance genes in addition to mcr-1, including 2 carbapenemases, blaNDM-1 and blaOXA-232. QS17-0161 carried 13 antibiotic resistance genes in addition to mcr-1, including the extended-spectrum ß-lactamase blaCTX-M-55. Both isolates carried multiple plasmids, but mcr-1 was located alone on highly similar 33.9 Kb IncX4 plasmids in both isolates. The IncX4 plasmid shared considerable homology to other mcr-1-containing IncX4 plasmids. This is the first report of a clinical K. pneumoniae strain from Thailand carrying mcr-1 as well as the first strain to simultaneously carry mcr-1 and multiple carbapenemase genes (QS17-0029). The identification and characterization of these isolates serves to highlight the urgent need for continued surveillance and intervention in Southeast Asia, where extensively drug-resistant pathogens are being increasingly identified in hospital-associated infections.

Bacillus anthracis Peptidoglycan Integrity Is Disrupted by the Chemokine CXCL10 through the FtsE/X Complex.

The antimicrobial activity of the chemokine CXCL10 against vegetative cells of Bacillus anthracis occurs via both bacterial FtsE/X-dependent and-independent pathways. Previous studies established that the FtsE/X-dependent pathway was mediated through interaction of the N-terminal region(s) of CXCL10 with a functional FtsE/X complex, while the FtsE/X-independent pathway was mediated through the C-terminal α-helix of CXCL10. Both pathways result in cell lysis and death of B. anthracis. In other bacterial species, it has been shown that FtsE/X is involved in cellular elongation though activation of complex-associated peptidoglycan hydrolases. Thus, we hypothesized that the CXCL10-mediated killing of vegetative cells of B. anthracis through the FtsE/X-dependent pathway resulted from the disruption of peptidoglycan processing. Immunofluorescence microscopy studies using fluorescent peptidoglycan probes revealed that incubation of B. anthracis Sterne (parent) strain with CXCL10 or a C-terminal truncated CXCL10 (CTTC) affected peptidoglycan processing and/or incorporation of precursors into the cell wall. B. anthracis ΔftsX or ftsE(K123A/D481N) mutant strains, which lacked a functional FtsE/X complex, exhibited little to no evidence of disruption in peptidoglycan processing by either CXCL10 or CTTC. Additional studies demonstrated that the B. anthracis parent strain exhibited a statistically significant increase in peptidoglycan release in the presence of either CXCL10 or CTTC. While B. anthracis ΔftsX strain showed increased peptidoglycan release in the presence of CXCL10, no increase was observed with CTTC, suggesting that the FtsE/X-independent pathway was responsible for the activity observed with CXCL10. These results indicate that FtsE/X-dependent killing of vegetative cells of B. anthracis results from a loss of cell wall integrity due to disruption of peptidoglycan processing and suggest that FtsE/X may be an important antimicrobial target to study in the search for alternative microbial therapeutics.

CXCL10 Acts as a Bifunctional Antimicrobial Molecule against Bacillus anthracis.

UNLABELLED: Bacillus anthracis is killed by the interferon-inducible, ELR(-) CXC chemokine CXCL10. Previous studies showed that disruption of the gene encoding FtsX, a conserved membrane component of the ATP-binding cassette transporter-like complex FtsE/X, resulted in resistance to CXCL10. FtsX exhibits some sequence similarity to the mammalian CXCL10 receptor, CXCR3, suggesting that the CXCL10 N-terminal region that interacts with CXCR3 may also interact with FtsX. A C-terminal truncated CXCL10 was tested to determine if the FtsX-dependent antimicrobial activity is associated with the CXCR3-interacting N terminus. The truncated CXCL10 exhibited antimicrobial activity against the B. anthracis parent strain but not the ΔftsX mutant, which supports a key role for the CXCL10 N terminus. Mutations in FtsE, the conserved ATP-binding protein of the FtsE/X complex, resulted in resistance to both CXCL10 and truncated CXCL10, indicating that both FtsX and FtsE are important. Higher concentrations of CXCL10 overcame the resistance of the ΔftsX mutant to CXCL10, suggesting an FtsX-independent killing mechanism, likely involving its C-terminal α-helix, which resembles a cationic antimicrobial peptide. Membrane depolarization studies revealed that CXCL10 disrupted membranes of the B. anthracis parent strain and the ΔftsX mutant, but only the parent strain underwent depolarization with truncated CXCL10. These findings suggest that CXCL10 is a bifunctional molecule that kills B. anthracis by two mechanisms. FtsE/X-dependent killing is mediated through an N-terminal portion of CXCL10 and is not reliant upon the C-terminal α-helix. The FtsE/X-independent mechanism involves membrane depolarization by CXCL10, likely because of its α-helix. These findings present a new paradigm for understanding mechanisms by which CXCL10 and related chemokines kill bacteria. IMPORTANCE: Chemokines are a class of molecules known for their chemoattractant properties but more recently have been shown to possess antimicrobial activity against a wide range of Gram-positive and Gram-negative bacterial pathogens. The mechanism(s) by which these chemokines kill bacteria is not well understood, but it is generally thought to be due to the conserved amphipathic C-terminal α-helix that resembles cationic antimicrobial peptides in charge and secondary structure. Our present study indicates that the interferon-inducible, ELR(-) chemokine CXCL10 kills the Gram-positive pathogen Bacillus anthracis through multiple molecular mechanisms. One mechanism is mediated by interaction of CXCL10 with the bacterial FtsE/X complex and does not require the presence of the CXCL10 C-terminal α-helix. The second mechanism is FtsE/X receptor independent and kills through membrane disruption due to the C-terminal α-helix. This study represents a new paradigm for understanding how chemokines exert an antimicrobial effect that may prove applicable to other bacterial species.