Genome characterization of the novel lytic phage vB_AbaAut_ChT04 and the antimicrobial activity of its lysin peptide against Acinetobacter baumannii isolates from different time periods.

Acinetobacter baumannii is an important opportunistic pathogen, usually associated with immunocompromised individuals with a prolonged period of stay in a hospital. Currently, the incidence of multi-drug resistant A. baumannii (MDR-AB) and extensively drug-resistant A. baumannii (XDR-AB) is increasing rapidly in Thailand, mirroring the trend worldwide. Novel therapeutic approaches for the treatment of A. baumannii infection using bacteriophages are being evaluated, and the use of phage-derived peptides is being tested as alternative approach to fighting infection. In this study, we isolated and determined the biological features of a lytic A. baumannii phage called vB_AbaAut_ChT04 (vChT04). The vChT04 phage was classified as a member of the family Autographiviridae of the class Caudoviricetes. It showed a short latent period (10 min) and a large burst size (280 PFU cell-1), and it was able to infect 52 out of 150 clinical MDR-AB strains tested (34.67%). Most of the phage-sensitive strains were A. baumannii strains that had been isolated during the same year that the phage was isolated. The phage showed activity across a broad pH (pH 5.0-8.0) and temperature (4-37°C) range. Whole-genome analysis revealed that the vChT04 genome comprises 41,158 bp with a 39.3% GC content and contains 48 open reading frames (ORFs), 28 of which were assigned putative functions based on homology to previously identified phage genes. Comparative genomic analysis demonstrated that vChT04 had the highest similarity to phage vB_AbaP_WU2001, which was isolated in the southern part of Thailand. An endolysin gene found in the vChT04 genome was used to synthesize an antimicrobial peptide (designated as PLysChT04) and its antimicrobial activity was evaluated using minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays. The MIC and MBC values of peptide PLysChT04 against MDR-AB and XDR-AB were 312.5-625 µg/mL, and it was able to inhibit both phage-susceptible and phage-resistant isolates collected over different time periods. PLysChT04 showed good efficacy in killing drug-resistant A. baumannii and other bacterial strains, and it is a promising candidate for development as an alternative therapeutic agent targeting A. baumannii infections.

Molecular Characteristics of Methicillin-Resistant Staphylococci Clinical Isolates from a Tertiary Hospital in Northern Thailand.

Methicillin-resistant staphylococci are now recognized as a major cause of infectious diseases, particularly in hospitals. Molecular epidemiology is important for prevention and control of infection, but little information is available regarding staphylococcal infections in Northern Thailand. In the present study, we examined antimicrobial susceptibility patterns, detection of antimicrobial resistance genes, and SCCmec types of methicillin-resistant S. aureus (MRSA) and methicillin-resistant coagulase-negative staphylococci (MR-CoNS) isolated from patients in a hospital in Northern Thailand. The species of MRSA and MR-CoNS were identified using combination methods, including PCR, MALDI-TOF-MS, and tuf gene sequencing. The susceptibility pattern of all isolates was determined by the disk diffusion method. Antimicrobial resistance genes, SCCmec types, and ST239 were characterized using single and multiplex PCR. ST239 was predominant in MRSA isolates (10/23). All MR-CoNS (N=31) were identified as S. haemolyticus (N=18), S. epidermidis (N=3), S. cohnii (N=3), S. capitis (N=6), and S. hominis (N=1). More than 70% of MRSA and MR-CoNS were resistant to cefoxitin, penicillin, oxacillin, erythromycin, clindamycin, gentamicin, and ciprofloxacin. In MRSA isolates, the prevalence of ermA (78.3%) and ermB (73.9%) genes was high compared to that of the ermC gene (4.3%). In contrast, ermC (87.1%) and qacA/B genes (70.9%) were predominant in MR-CoNS isolates. SCCmec type III was the dominant type of MRSA (13/23), whereas SCCmec type II was more present in S. haemolyticus (10/18). Ten MRSA isolates with SCCmec type III were ST239, which is the common type of MRSA in Asia. This finding provides useful information for a preventive health strategy directed against methicillin-resistant staphylococcal infections.

Characterization and Detection of Endolysin Gene from Three Acinetobacter baumannii Bacteriophages Isolated from Sewage Water.

Acinetobacter baumannii is an opportunistic pathogen that exists in hospital environments. The emergence of multidrug resistant A. baumannii (MDRAB) has been reported worldwide. It is necessary to find a novel and effective treatment for MDRAB infection. In this study, three bacteriophages, designated as ØABP-01, ØABP-02 and ØABP-04 were selected for analysis. Transmission electron microscopy showed that bacteriophage ØABP-01 belonged to the Podoviridae family and bacteriophage ØABP-02 and ØABP-04 are classified into the family Myoviridae. ØABP-01 had the widest host range. ØABP-01, ØABP-02 and ØABP-04 exhibited a latent period of 15, 20 and 20 min. The burst sizes of the three bacteriophages were 110, 120 and 150 PFU/cell. DNA restriction analysis using EcoRI, HindIII, PstI, SphI, BamHI and SmaI showed different DNA fragment patterns between the three bacteriophages. ØABP-01 and ØABP-04 was positive for the endolysin gene as determined by PCR. In conclusion, bacteriophage ØABP-01 showed broad host-specificity, good lytic activity and a short latency period, making it an appropriate candidate for studying the control and diagnosis associated with MDRAB infections.

Genomic Landscape Reveals Chromosomally-Mediated Antimicrobial Resistome and Virulome of a High-Risk International Clone II Acinetobacter baumannii AB073 from Thailand.

This study utilized integrative bioinformatics' tools together with phenotypic assays to understand the whole-genome features of a carbapenem-resistant international clone II Acinetobacter baumannii AB073. Overall, we found the isolate to be resistant to seven antibiotic classes, penicillins, ß-lactam/ß-lactamase inhibitor combinations, cephalosporins, carbapenems, aminoglycosides, fluoroquinolones, and folate pathway antagonists. These resistance phenotypes are related to various chromosomal-located antibiotic resistance determinants involved in different mechanisms such as reduced permeability, antibiotic target protection, antibiotic target alteration, antibiotic inactivation, and antibiotic efflux. IC2 A. baumannii AB073 could not transfer antibiotic resistance by conjugation experiments. Likewise, mobilome analysis found that AB073 did not carry genetic determinants involving horizontal gene transfer. Moreover, this isolate also carried multiple genes associated with the ability of iron uptake, biofilm formation, immune invasion, virulence regulations, and serum resistance. In addition, the genomic epidemiological study showed that AB073-like strains were successful pathogens widespread in various geographic locations and clinical sources. In conclusion, the comprehensive analysis demonstrated that AB073 contained multiple genomic determinants which were important characteristics to classify this isolate as a successful international clone II obtained from Thailand.

Genomic relatedness and dissemination of bla NDM-5 among Acinetobacter baumannii isolated from hospital environments and clinical specimens in Thailand.

Background: Acinetobacter baumannii (A. baumannii) is an important cause of nosocomial infection, especially in intensive care units (ICUs). It has the propensity to tolerate various environments and multiple classes of antibiotics. Our study aimed to characterize the comparative genomes of A. baumannii from hospital environments and clinical isolates. Methods: Clinical and environmental A. baumannii isolates were collected from a university hospital. Antibiotic susceptibility testing was performed, antibiotic resistance genes (ARGs) were characterized, and repetitive element palindromic-PCR (rep-PCR) typing was performed. Eight representative A. baumannii isolated from environmental and clinical samples from the same wards were selected for whole-genome sequencing (WGS) using the Illumina platform. Results: A total of 106 A. baumannii isolates were obtained from 312 hospital environmental samples. A high percentage of samples with A. baumannii colonization were detected from AMBU bags (77.9%), followed by bedrails (66.7%) and suction tubes (66.7%). We found that 93.4% of the environmental isolates were multidrug-resistant A. baumannii (MDRAB), and 44.7% were extremely drug-resistant A. baumannii (XDRAB). bla OXA-23 bla NDM, and bla OXA-58 were present in 80.2%, 78.3%, and 0.9% of all isolates, respectively. Sixty-one A. baumannii isolates were collected from patient specimens in the same ward. Among all A. baumannii clinical isolates, MDRAB and XDRAB accounted for 82% and 55.7%, respectively. The most dominant ARGs identified was bla OXA-23 (80.3%), followed by bla NDM (55.7%). The genetic diversity of all isolates using rep-PCR could be divided into 33 genotypes. The genome size of eight A. baumannii ranged from 3.78-4.01 Mb. We found six of eight strains to be bla NDM-5-harboring A. baumannii. Mobile genetic elements (MGEs), such as integron1 (intl1), located upstream of bla NDM-5 were observed. The phylogenomic relationship of the core and pan genomes as well as the single nucleotide polymorphism (SNP) count matrix revealed the genetic similarity of A. baumannii environmental and clinical strains obtained from the same ward. Conclusion: This study confirmed that A. baumannii colonized in hospital environments were the main reservoir of nosocomial infection and provides critical information to guide the control of A. baumannii infection.

Prevalence and virulence genes of Staphylococcus aureus from food contact surfaces in Thai restaurants.

Background: Staphylococcus aureus is one of the most common pathogens responsible for food poisoning due to its ability to produce staphylococcal enterotoxin (SE). S. aureus can form biofilms on the surfaces of food processing devices, enabling the distribution of SE on foods through cross-contamination events. Thailand is known for its exotic cuisine, but there is no data on the prevalence of SE-harboring S. aureus in restaurants in Thailand. Methods: In this study, we conducted surface swabs on surfaces of kitchen utensil that come into contact with food and on the hands of food handlers working in restaurants in the north part of Thailand. Isolated S. aureus was investigated for biofilm formation, virulence, and SE genes. Results: Two hundred S. aureus were isolated from 650 samples. The highest prevalence of S. aureus contamination was detected on the hands of food handlers (78%), followed by chopping boards (26%), plates (23%), knives (16%), spoons (13%), and glasses (5%). All of them were methicillin-sensitive S. aureus (MSSA) and the mecA gene was not present in any strains. Biofilm formation was detected using the CRA method, and 49 (24.5%) were identified as biofilm-producing strains, with the hands of food handlers identified as the primary source of biofilm-producing strains. The prevelence of biofilm-related adhesion genes detected were: icaAD (13%), fnbA (14.5%), cna (6.5%), and bap (0.5%). Two classical enterotoxin genes, sec and sed, were also found in four and six of the S. aureus isolates, respectively, from hands and utensils. Conclusion: The highest prevelence of S. aureus was detected on the hands of food handlers. S. aureus strains with biofilm and enterotoxin production abilities were discovered on food contact surfaces and the hands of food handlers, implying significant risk of food contamination from these sources that could be harmful to consumers. To avoid cross-contamination of food with food contact items, the food handlers' hands should be properly washed, and all food preparation equipment should be thoroughly cleaned.

Antibacterial activity of vB_AbaM_PhT2 phage hydrophobic amino acid fusion endolysin, combined with colistin against Acinetobacter baumannii.

Phage lytic enzymes are promising antimicrobial agents. In this study, an endolysin derived from vB_AbaM_PhT2 (vPhT2), was identified. This endolysin represented the conserved lysozyme domain. Recombinant endolysin (lysAB- vT2) and hydrophobic fusion endolysin (lysAB-vT2-fusion) were expressed and purified. Both endolysins showed lytic activity against bacterial crude cell wall of Gram-negative bacteria. The MIC of lysAB-vT2-fusion was 2 mg/ml corresponding to 100 µM, while the MIC of lysAB-vT2 was more than 10 mg/ml (400 µM). Combination of lysAB-vT2-fusion with colistin, polymyxin B or copper was synergistic against A. baumannii (FICI value as 0.25). Antibacterial activity of lysAB-vT2-fusion plus colistin at the fractional inhibitory concentrations (FICs) revealed that it can inhibit Escherichia coli, Klebsiella pneumoniae and various strains of extremely drug-resistant A. baumannii (XDRAB) and phage resistant A. baumannii. The lysAB- vT2-fusion still retained its antibacterial activity after incubating the enzyme at 4, 20, 40 and 60 °C for 30 min. The lysAB-vT2-fusion could inhibit the mature biofilm, and incubation of lysAB-vT2-fusion with T24 human cells infected with A. baumannii led to a partial reduction of LDH release from T24 cells. In summary, our study highlights the antimicrobial ability of engineered lysAB-vT2-fusion endolysin, which can be applied for the control of A. baumannii infection.

Genomic analysis uncovers laccase-coding genes and biosynthetic gene clusters encoding antimicrobial compounds in laccase-producing Acinetobacter baumannii.

Laccases are multicopper oxidase family enzymes that can oxidize various substrates. In this study, we isolated laccase-producing Acinetobacter spp. from the environment, and one isolate of laccase-producing Acinetobacter baumannii, designated NI-65, was identified. The NI-65 strain exhibited constitutive production of extracellular laccase in a crude extract using 2,6-dimethoxyphenol as a substrate when supplemented with 2 mM CuSO4. Whole-genome sequencing of the NI-65 strain revealed a genome size of 3.6 Mb with 3,471 protein-coding sequences. The phylogenetic analysis showed high similarity to the genome of A. baumannii NCIMB8209. Three laccase proteins, PcoA and CopA, that belong to bacterial CopA superfamilies, and LAC-AB, that belongs to the I-bacterial bilirubin oxidase superfamily, were identified. These proteins were encoded by three laccase-coding genes (pcoA, copA, and lac-AB). The lac-AB gene showed a sequence similar to that of polyphenol oxidase (PPO). Gene clusters encoding the catabolized compounds involved in the utilization of plant substances and secondary metabolite biosynthesis gene clusters encoding antimicrobial compounds were identified. This is the first report of whole-genome sequencing of laccase-producing A. baumannii, and the data from this study help to elucidate the genome of A. baumannii to facilitate its application in synthetic biology for enzyme production.

Insights into mobile genetic elements and the role of conjugative plasmid in transferring aminoglycoside resistance in extensively drug-resistant Acinetobacter baumannii AB329.

Acinetobacter baumannii is a major cause of nosocomial infection, and the incidence of extensively drug-resistant A. baumannii (XDRAB) infections has dramatically increased worldwide. In this study, we aimed to explore the complete genome sequence of XDRAB 329, ST1166/98 (Oxford/Pasteur), which is an outbreak clone from a hospital in Thailand. Whole-genome sequencing (WGS) was performed using short-read Illumina and long-read PacBio sequencing, and a conjugation assay of its plasmid was performed. The complete genome sequence of A. baumannii AB329 revealed a circular chromosome 3,948,038 bp in length with 39% GC content. Antibiotic resistance genes (ARGs), including beta-lactam resistance (bla OXA-51, bla ADC-25, bla OXA-23, bla TEM-1D), aminoglycoside resistance (aph(3')-Ia, aph(3″)-Ib, aph(6)-Id, armA), tetracycline resistance (tet(B), tet (R)), macrolide resistance (mph(E), msr(E)), and efflux pumps, were found. Mobile genetic elements (MGEs) analysis of A. baumannii AB329 revealed two plasmids (pAB329a and pAB329b), three prophages, 19 genomic islands (GIs), and 33 insertion sequences (ISs). pAB329a is a small circular plasmid of 8,731 bp, and pAB329b is a megaplasmid of 82,120 bp. aph(3')-VIa was detected in pAB329b, and a major facilitator superfamily (MFS) transporter was detected in the prophage. Acinetobacter baumannii resistance island 4 (AbaR4) harboring tetracycline and aminoglycoside resistance was detected in the genome of A. baumannii AB329. pAB329b, which belongs to Rep-type GR6 (plasmid lineage LN_1), is a conjugative plasmid with the ability to transfer an aminoglycoside resistance gene to sodium azide-resistant A. baumannii. This study provides insights into the features of the MGEs of XDRAB, which are the main reservoir and source of dissemination of ARGs.

Comparative genome analysis of Escherichia coli bacteriophages isolated from sewage and chicken meat.

Escherichia coli, a bacterium that causes severe foodborne diseases, is transmitted to humans primarily through the consumption of contaminated foods. These foodborne pathogens are causing a public health problem that requires alternative control approaches, such as bacteriophage (phage) biocontrol. In this study, we characterized vB_EcoM_Tw01 (vTw01) isolated from sewage and vB_EcoM_Tcm05 (vTcm05) isolated from chicken meat. Both vTw01 and vTcm05 were assigned to the family Myoviridae based on their morphology, with the former exhibiting a narrow host range with low minimum inhibitory multiplicity of infection (miMOI), and the latter exhibiting a broad host range with high miMOI. The latent periods of these phages were 20 and 30 min for vTw01 and vTcm05, while the burst sizes were ∼140 and ∼300 PFU/cell, respectively. They were relatively stable over a wide range of pH values and temperatures. The bioinformatics analysis of the genomic sequence suggests that vTw01 and vTcm05 have double-stranded DNA with genome sizes of 170,107 bp and 149,059 bp, respectively. Bacteriophage encoded enzymes, such as tail-lysozyme, spanin Rz, holin, cell wall hydrolase (CWH), and endolysin, were identified in the genome of both phages. In conclusion, this study investigated the morphological, physiological, and genomic features of two E. coli phages isolated from different sources. It was confirmed that these phages and their enzymes can serve as potential candidates for phage biocontrol.

Genome analysis of secondary metabolite­biosynthetic gene clusters of Photorhabdus akhurstii subsp. akhurstii and its antibacterial activity against antibiotic-resistant bacteria.

Xenorhabdus and Photorhabdus can produce a variety of secondary metabolites with broad spectrum bioactivity against microorganisms. We investigated the antibacterial activity of Xenorhabdus and Photorhabdus against 15 antibiotic-resistant bacteria strains. Photorhabdus extracts had strong inhibitory the growth of Methicillin-resistant Staphylococcus aureus (MRSA) by disk diffusion. The P. akhurstii s subsp. akhurstii (bNN168.5_TH) extract showed lower minimum inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC). The interaction between either P. akhurstii subsp. akhurstii (bNN141.3_TH) or P. akhurstii subsp. akhurstii (bNN168.5_TH) or P. hainanensis (bNN163.3_TH) extract in combination with oxacillin determined by checkerboard assay exhibited partially synergistic interaction with fractional inhibitory concentration index (FICI) of 0.53. Time-killing assay for P. akhurstii subsp. akhurstii (bNN168.5_TH) extract against S. aureus strain PB36 significantly decreased cell viability from 105 CFU/ml to 103 CFU/ml within 30 min (P < 0.001, t-test). Transmission electron microscopic investigation elucidated that the bNN168.5_TH extract caused treated S. aureus strain PB36 (MRSA) cell membrane damage. The biosynthetic gene clusters of the bNN168.5_TH contained non-ribosomal peptide synthetase cluster (NRPS), hybrid NRPS-type l polyketide synthase (PKS) and siderophore, which identified potentially interesting bioactive products: xenematide, luminmide, xenortide A-D, luminmycin A, putrebactin/avaroferrin and rhizomide A-C. This study demonstrates that bNN168.5_TH showed antibacterial activity by disrupting bacterial cytoplasmic membrane and the draft genome provided insights into the classes of bioactive products. This also provides a potential approach in developing a novel antibacterial agent.

Insight into Molecular Epidemiology, Antimicrobial Resistance, and Virulence Genes of Extensively Drug-Resistant Acinetobacter baumannii in Thailand.

Extensively drug-resistant Acinetobacter baumannii (XDR-AB) is a major threat to public health worldwide. A retrospective study for 27 XDR-AB isolates from four tertiary hospitals in Thailand was conducted. Beta-lactamase and virulence genes were characterized by PCR. The blaADC, blaOXA-51, and blaOXA-23 were detected in all isolates, whereas blaPER-1 and blaNDM-1 genes were present in 7.4% and 3.7% of isolates. All isolates had virulence genes, including genes in iron acquisition system, biofilm formation and secretion systems. The plasmids in XDR-AB belonged to GR2 (100%), GR6 (40.7%), and GR1 (7.4%). Multilocus sequence typing sequence types (STs) were further investigated. The data demonstrated that XDR-AB isolates had nine STs: ST195 (n = 4), ST208 (n = 4), ST368 (n = 1), ST451 (n = 5), ST457 (n = 2), ST1947 (n = 1), ST1166 (n = 7), including two novel STs namely ST1682 (n = 2) and ST1684 (n = 1). We observed that the majority ST1166 (25.9%) was associated with the prevalence of GR2 and GR6 plasmids and traU virulence gene. Genome-based single nucleotide polymorphism phylogenetic analysis of the isolates with two novel ST types indicated that the two isolates belonged to the international clone II (IC2) within the same cluster. In conclusion, our data showed the dissemination of XDR-AB isolates harbored virulence genes and antibiotic resistance genes among four hospitals in Thailand. The results highlighted the difficulty posed for the empirical treatment of the patients with the A. baumannii infection.

Essential Gene Clusters Involved in Copper Tolerance Identified in Acinetobacter baumannii Clinical and Environmental Isolates.

Copper is widely used as antimicrobial in agriculture and medicine. Copper tolerance mechanisms of pathogenic bacteria have been proven to be required for both copper tolerance and survival during bacterial infections. Here, we determined both copper-tolerant phenotype and genotype in A. baumannii originated from clinical and environmental samples. Using copper susceptibility testing, copper-tolerant A. baumannii could be found in both clinical and environmental isolates. Genotypic study revealed that representative copper-related genes of the cluster A (cueR), B (pcoAB), and D (oprC) were detected in all isolates, while copRS of cluster C was detected in only copper-tolerant A. baumannii isolates. Moreover, we found that copper-tolerant phenotype was associated with amikacin resistance, while the presence of copRS was statistically associated with blaNDM-1. We chose the A. baumannii strain AB003 as a representative of copper-tolerant isolate to characterize the effect of copper treatment on external morphology as well as on genes responsible for copper tolerance. The morphological features and survival of A. baumannii AB003 were affected by its exposure to copper, while whole-genome sequencing and analysis showed that it carried fourteen copper-related genes located on four clusters, and cluster C of AB003 was found to be embedded on genomic island G08. Transcriptional analysis of fourteen copper-related genes identified in AB003 revealed that copper treatment induced the expressions of genes of clusters A, B, and D at the micromolar level, while genes of cluster C were over-expressed at the millimolar levels of copper. This study showed that both clinical and environmental A. baumannii isolates have the ability to tolerate copper and carried numerous copper tolerance determinants including intrinsic copper tolerance (clusters A, B, and D) and acquired copper tolerance (cluster C) that could respond to copper toxicity. Our evidence suggests that we need to reconsider the use of copper in hospitals and other medical environments to prevent the selection and spread of copper-tolerant organisms.

Genomic analysis reveals high virulence and antibiotic resistance amongst phage susceptible Acinetobacter baumannii.

In this study, we examined the association between antimicrobial resistance, CRISPR/Cas systems and virulence with phage susceptibility in Acinetobacter baumannii and investigated draft genomes of phage susceptible multidrug resistant A. baumannii strains from Thailand. We investigated 230 A. baumannii strains using 17 lytic A. baumannii phages and the phage susceptibility was 46.5% (107/230). Phage susceptibility was also associated with resistance to numerous antibiotics (p-value < 0.05). We also found association between biofilm formation and the presence of ompA gene among phage susceptible A. baumannii strains (p-value < 0.05). A. baumannii isolates carrying cas5 or combinations of two or three other cas genes, showed a significant increase in phage resistance. Whole-genome sequences of seven phage susceptible A. baumannii isolates revealed that six groups of antibiotic resistance genes were carried by all seven phage susceptible A. baumannii. All strains carried biofilm associated genes and two strains harbored complete prophages, acquired copper tolerance genes, and CRISPR-associated (cas) genes. In conclusion, our data exhibits an association between virulence determinants and biofilm formation among phage susceptible A. baumannii strains. These data help to understand the bacterial co-evolution with phages.

Investigating Bacteriophages Targeting the Opportunistic Pathogen Acinetobacter baumannii.

The multi-drug resistance of the opportunistic pathogen Acinetobacter baumannii is of growing concern, with many clinical isolates proving to be resistant to last resort as well as front line antibiotic treatments. The use of bacteriophages is an attractive alternative to controlling and treating this emerging nosocomial pathogen. In this study, we have investigated bacteriophages collected from hospital wastewater in Thailand and we have explored their activity against clinical isolates of A. baumannii. Bacteriophage vB_AbaM_PhT2 showed 28% host range against 150 multidrug resistant (MDR) isolates and whole genome sequencing did not detect any known virulence factors or antibiotic resistance genes. Purified vB_AbaM_PhT2 samples had endotoxin levels below those recommended for preclinical trials and were not shown to be directly cytotoxic to human cell lines in vitro. The treatment of human brain and bladder cell lines grown in the presence of A. baumannii with this bacteriophage released significantly less lactate dehydrogenase compared to samples with no bacteriophage treatment, indicating that vB_AbaM_PhT2 can protect from A. baumannii induced cellular damage. Our results have also indicated that there is synergy between this bacteriophage and the end line antibiotic colistin. We therefore propose bacteriophage vB_AbaM_PhT2 as a good candidate for future research and for its potential development into a surface antimicrobial for use in hospitals.

Biofilm Formation of Methicillin-resistant Coagulase-Negative Staphylococci Isolated from Clinical Samples in Northern Thailand.

BACKGROUND: Methicillin-resistant coagulase-negative staphylococci (MR-CoNS) are multidrug-resistant bacteria that are difficult to treat because of their ability to form biofilms. OBJECTIVES: In the present study, we evaluated the antibiotic-resistant phenotypes, biofilm-forming ability, and biofilm associated genes of 55 clinical MR-CoNS isolates obtained from two hospitals in Thailand. MATERIALS AND METHODS: MALDI-TOF-MS and tuf gene sequencing were performed to determine the species of all isolates. Biofilm production was determined using Congo red agar (CRA) and the microtiter plate (MTP) assay. Biofilm-associated genes were characterized using polymerase chain reaction (PCR). RESULTS: Among the 55 MR-CoNS isolates, five species were identified as Staphylococcus haemolyticus (34.5%), Staphylococcus epidermidis (32.7%), Staphylococcus capitis (18.2%), Staphylococcus cohnii (9.1%), and Staphylococcus hominis (5.5%). The antimicrobial susceptibility pattern of MR-CoNS isolates indicated high resistance to cefoxitin (100%), penicillin (98.2%), erythromycin (96.4%), ciprofloxacin (67.3%), sulfamethoxazole/trimethoprim (67.3%), gentamicin (67.3%), and clindamycin (63.6%). All the isolates were susceptible to vancomycin and linezolid. The biofilm production was detected in 87.3% isolates through the CRA method and in 38.1% isolates through the MTP assay. The prevalence rates of icaAD, bap, fnbA, and cna were 18.2%, 12.7%, 47.3%, and 27.3%, respectively. There were significant differences in the presence of these biofilm-associated genes among the MR-CoNS isolates. Moreover, quantitative biofilm formation was significantly different among MR-CoNS species. CONCLUSION: The present study revealed that biofilm-associated genes are important for biofilm biomass in MR-CoNS isolates, and the findings of this study are essential for finding new strategies to control biofilm formation and prevent the spread of MR-CoNS infectious diseases.

Molecular Characterization of Colistin-Resistant Escherichia coli Isolated from Chickens: First Report from Nepal.

Dissemination of mcr-1 encoding colistin resistance in Gram-negative bacteria has created critical situation in poultry, livestock farming, and public health. In Nepal, for the first time, we initiated surveillance of colistin-resistant Escherichia coli in broilers from seven different chicken farms. A total of 324 cloacal swabs were collected and 118 E. coli were isolated, of which 27 (22.8%) were colistin resistance all harboring mcr-1 gene, but lacking ISApI1. Colistin-resistant isolates were characterized by antibiotic susceptibility testing, detecting antibiotic resistance genes, phylogenetic analysis, and plasmid replicon typing. These isolates belonged to the phylo-group A (70.37%) and phylo-group D (29.63%). In addition, most isolates (>80%) were resistant to ciprofloxacin, tetracycline, and sulfamethoxazole-trimethoprim. As much as 3 of the 27 mcr-1 encoding isolates were confirmed as extended-spectrum ß-lactamase (ESBL) producer, all 3 isolates carrying blaCTX-M gene. We performed the conjugation experiment to check transferability of mcr-1, tet, and blaCTX-M genes, and only two donors were found to have transferred resistance to ticarcillin. The transfer of colistin and tetracycline resistance was not detected, which suggests the chromosomal location of mcr-1 and tet genes. The prevalence of Inc K/B and Inc I1 was 96.3% and 81.48%, respectively. This study shows the co-existence of mcr-1 with tet, sul, qnr, dfr, and blaCTX-M genes and dissemination of these resistant isolates in Nepalese chicken farms, which may pose huge threat to the livestock, especially chickens, and public health in Nepal.

The emergence of colistin-resistant Escherichia coli in chicken meats in Nepal.

The emergence and dissemination of colistin resistance among Gram-negative bacteria is a global problem. We initiated a surveillance of colistin-resistant and -susceptible Escherichia coli in raw meats from chicken in Nepal. A total of 180 meat samples were collected; from these, 60 E. coli strains were isolated (33.33%), of which 16 (26.66%) were colistin-resistant and harboured the mcr-1 gene. All isolates were characterised by antibiotic susceptibility testing, the presence of antibiotic resistance genes, phylogenetic analysis and plasmid replicon typing. Most of the colistin-resistant E. coli had the antibiotic resistant pattern CIP/CN/SXT/TE (43.75%). Coexistence of tet, qnr, sul and dfr genes was detected in both colistin-resistant and -susceptible E. coli. Most colistin-resistant E. coli strains belonged to phylogroup C, whereas 10% of isolates belonged to phylogroup D. Inc FIB was the dominant plasmid Inc type in the isolates. Dissemination of antibiotic-resistant E. coli in raw meats is a public health concern in Nepal and requires further investigation to ascertain the sources of contamination.

Acquisition and transfer of antibiotic resistance genes in association with conjugative plasmid or class 1 integrons of Acinetobacter baumannii.

Conjugation is a type of horizontal gene transfer (HGT) that serves as the primary mechanism responsible for accelerating the spread of antibiotic resistance genes in Gram-negative bacteria. The present study aimed to elucidate the mechanisms underlying the conjugation-mediated gene transfer from the extensively drug-resistant Acinetobacter baumannii (XDR-AB) and New Delhi Metallo-beta-lactamase-1-producing Acinetobacter baumannii (NDM-AB) to environmental isolates of Acinetobacter spp. Conjugation experiments demonstrated that resistance to ticarcillin and kanamycin could be transferred from four donors to two sodium azide-resistant A. baumannii strains, namely, NU013R and NU015R. No transconjugants were detected on Mueller-Hinton Agar (MHA) plates containing tetracycline. Plasmids obtained from donors as well as successful transconjugants were characterized by PCR-based replicon typing and S1-nuclease pulsed-field gel electrophoresis (S1-PFGE). Detection of antibiotic resistance genes and integrase genes (int) was performed using PCR. Results revealed that the donor AB364 strain can transfer the blaOXA-23 and blaPER-1 genes to both recipients in association with int1. A 240-kb plasmid was successfully transferred from the donor AB364 to recipients. In addition, the aphA6 and blaPER-1 genes were co-transferred with the int1 gene from the donor strains AB352 and AB405. The transfer of a 220-kb plasmid from the donors to recipient was detected. The GR6 plasmid containing the kanamycin resistance gene (aphA6) was successfully transferred from the donor strain AB140 to both recipient strains. However, the blaNDM-1 and tet(B) genes were not detected in all transconjugants. Our study is the first to demonstrate successful in vitro conjugation, which indicated that XDR-AB contained combination mechanisms of the co-transfer of antimicrobial resistance elements with integron cassettes or with the plasmid group GR6. Thus, conjugation could be responsible for the emergence of new types of antibiotic-resistant strains.

Dissemination of blaOXA-23, blaOXA-24, blaOXA-58, and blaNDM-1 Genes of Acinetobacter baumannii Isolates from Four Tertiary Hospitals in Thailand.

Acinetobacter baumannii is a major threat to public health due to the emergence and dissemination of antibiotic-resistant strains. The purpose of this study was to determine the molecular epidemiology of antibiotic-resistant A. baumannii isolates collected from four tertiary hospitals in Thailand during the period November 2013-February 2015. We screened 339 A. baumannii, nonrepetitive clinical isolates to determine drug susceptibility. Among all isolates, we found that 7.9% was nondrug-resistant A. baumannii (NR-AB). Carbapenem-resistant A. baumannii (CR-AB) strains accounted for 84.9% of the total isolates, with extensively drug-resistant A. baumannii (XDR-AB) accounting for 7.9% of the total isolates. We further investigated class D carbapenemase genes using multiplex-PCR amplification and class B metallo-ß-lactamase genes, including blaIMP, blaVIM, and blaNDM-1 genes, using PCR and sequencing methods. We found that 300 (88.5%) isolates carried acquired class D carbapenemase genes, including blaOXA-23 (82.6%), blaOXA-24 (0.3%), and blaOXA-58 (6.5%). The genes blaIMP and blaVIM were not detected in any isolates. The blaNDM-1 was detected in 31 isolates from two hospitals (9.1%). All of the blaNDM-1-positive A. baumannii (NDM-AB) had ISAba125 sequences upstream of the blaNDM-1 gene. A coexistence of three resistance genes-blaOXA-23-blaOXA-58-blaNDM-1-was found in one isolate. A repetitive element palindromic-PCR (REP-PCR) revealed that all A. baumannii isolates were genetically diverse and could be divided into 33 genotypes. Only three genotypes were found to be predominant in all hospitals. Data from our study indicate the widespread emergence of multiple resistance determinants in A. baumannii isolates in Thailand, suggesting the need for more stringent infection control measures.