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Abstract The surge in mobile colistin-resistant genes (mcr) has become an increasing public health concern, especially in carbapenem-resistant Enterobacterales (CRE). Prospective surveillance was conducted to explore the genomic characteristics of clinical CRE isolates harbouring mcr in 2015–2020. In this study, we aimed to examine the genomic characteristics and phonotypes of mcr-8 and mcr-9 harbouring carbapenem-resistant K. pneumoniae complex (CRKpnC). Polymerase chain reaction test and genome analysis identified CRKpnC strain AMR20201034 as K. pneumoniae (CRKP) ST147 and strain AMR20200784 as K. quasipneumoniae (CRKQ) ST476, harbouring mcr-8 and mcr-9, respectively. CRKQ exhibited substitutions in chromosomal-mediated colistin resistance genes (pmrB, pmrC, ramA, and lpxM), while CRKP showed two substitutions in crrB, pmrB, pmrC, lpxM and lapB. Both species showed resistance to colistin, with minimal inhibitory concentrations of 8 µg/ml for mcr-8-carrying CRKP isolate and 32 µg/ml for mcr-9-carrying CRKQ isolate. In addition, CRKP harbouring mcr-8 carried bla NDM, while CRKQ harbouring mcr-9 carried bla IMP, conferring carbapenem resistance. Analysis of plasmid replicon types carrying mcr-8 and mcr-9 showed FIA-FII (96,575 bp) and FIB-HI1B (287,118 bp), respectively. In contrast with the plasmid carrying the carbapenemase genes, the CRKQ carried bla IMP-14 on an IncC plasmid, while the CRKP harboured bla NDM-1 on an FIB plasmid. This finding provides a comprehensive insight into another mcr-carrying CRE from patients in Thailand. The other antimicrobial-resistant genes in the CRKP were bla CTX-M-15, bla SHV-11, bla OXA-1, aac(6′)-Ib-cr, aph(3′)-VI, ARR-3, qnrS1, oqxA, oqxB, sul1, catB3, fosA, and qacE, while those detected in CRKQ were bla OKP-B-15, qnrA1, oqxA, oqxB, sul1, fosA, and qacE. This observation highlights the importance of strengthening official active surveillance efforts to detect, control, and prevent mcr-harbouring CRE and the need for rational drug use in all sectors.

Background and Aim: Bovine mastitis is an inflammation of the mammary gland of dairy cattle that causes economic losses due to poor quantity and quality of milk. The extensive or incorrect use of antibiotics has increased in the veterinary field, leading to the emergence of antibiotic-resistant pathogens worldwide. This study aimed to investigate bovine mastitis bacterial pathogens in Sakon Nakhon, Thailand. Materials and Methods: A total of 35 dairy farms were screened for clinical and subclinical mastitis using the California Mastitis Test and clinical examination. Polymerase chain reaction was used to characterize bacterial species-induced mastitis (380 isolates) in cattle and antimicrobial resistance genes, and disk diffusion and broth microdilution were used to characterize antimicrobial susceptibility. Results: The prevalence of Staphylococcus epidermidis (38.10%; 32/84)-induced mastitis in cattle was considerably high, followed by Streptococcus agalactiae (33.33%), Streptococcus uberis (25%), Klebsiella pneumoniae (8.33%), and Staphylococcus aureus (4.76%). In this study, Staphylococcus spp. isolates demonstrated 100% susceptibility to cefoxitin, and no antibiotic-resistance genes were identified. Tetracycline (TET) and macrolide-resistant genes of Streptococcus spp. revealed that tetM was predominant in 55.63% (79/142), followed by tetS + erm(B) (16.90%). Antibiotic susceptibility tests revealed the following resistance profiles to bacterial species: TET (85.92%), clindamycin (29.58%), erythromycin (15.49%), levofloxacin (14.08%), and penicillin (0%). Gram-negative bacterial isolates (K. pneumoniae [8.33%], Klebsiella variicola [2.38%], Klebsiella quasipneumoniae [1.19%], and Escherichia coli [1.19%]) were recovered and still susceptible to meropenem (100%), ceftazidime (97.06%), ceftriaxone (79.41%), and ciprofloxacin (79.41%). Conclusion: This result suggested that mastitis pathogens in this area were susceptible to most antimicrobials, with the exception of streptococci against TET. In this study, limited data were available including one from small-holder dairy farms and study only dairy farms in Sakon Nakhon, Thailand. So, more farms should be included in the future studies.

Staphylococcus epidermidis, a coagulase-negative staphylococcus, is a prevalent skin commensal that has increasingly been recognized as a significant pathogen, particularly in hospital environments, where it is associated with device-related infections. The emergence of multi-drug resistance and its ability to form biofilms complicate the clinical management of infections caused by this organism, posing a growing public health concern. This study aimed to investigate the nasal carriage of S. epidermidis among healthy young individuals and to analyze its antibiotic resistance patterns, resistance genes, and biofilm formation capabilities. Nasal swabs were collected from 40 undergraduate students at Mahasarakham University, Thailand, aged between 20 and 22 years. A total of 38 isolates were confirmed as S. epidermidis through both phenotypic and molecular characterization. Antibiotic susceptibility testing demonstrated resistance to various classes of antimicrobials, including beta-lactams, macrolides, and tetracyclines. Notably, five isolates exhibited methicillin resistance S. epidermidis (MRSE). Resistance genes, such as mecA, ermA, tetM, tetL, and tetK, were identified across the isolates, contributing to the observed resistance profiles. Biofilm formation assays revealed that most isolates displayed weak to moderate biofilm formation, with only one isolate demonstrating strong biofilm-forming capacity. Genetic analysis indicated a significant correlation between biofilm formation and the presence of the icaA gene, which is crucial for biofilm production. This study suggests the necessity for ongoing surveillance of nasal carriage of S. epidermidis to enhance understanding of its role in the dissemination of antimicrobial resistance and biofilm-associated infections, particularly within healthcare settings.

ABSTRACTStreptococcus suis is a major bacterial pathogen in pigs and an emerging zoonotic pathogen. Different S. suis serotypes exhibit diverse characteristics in population structure and pathogenicity. Surveillance data highlight the significance of S. suis serotype 4 (SS4) in swine streptococcusis, a pathotype causing human infections. However, except for a few epidemiologic studies, the information on SS4 remains limited. In this study, we investigated the population structure, pathogenicity, and antimicrobial characteristics of SS4 based on 126 isolates, including one from a patient with septicemia. We discovered significant diversities within this population, clustering into six minimum core genome (MCG) groups (1, 2, 3, 4, 7-2, and 7-3) and five lineages. Two main clonal complexes (CCs), CC17 and CC94, belong to MCG groups 1 and 3, respectively. Numerous important putative virulence-associated genes are present in these two MCG groups, and 35.00% (7/20) of pig isolates from CC17, CC94, and CC839 (also belonging to MCG group 3) were highly virulent (mortality rate ≥ 80%) in zebrafish and mice, similar to the human isolate ID36054. Cytotoxicity assays showed that the human and pig isolates of SS4 strains exhibit significant cytotoxicity to human cells. Antimicrobial susceptibility testing showed that 95.83% of strains isolated from our labs were classified as multidrug-resistant. Prophages were identified as the primary vehicle for antibiotic resistance genes. Our study demonstrates the public health threat posed by SS4, expanding the understanding of SS4 population structure and pathogenicity characteristics and providing valuable information for its surveillance and prevention.

Abstract This study aimed to determine the presence and characteristics of locally circulating strains of Streptococcus suis, the most important streptococcal pathogen in swine. Oral swab samples were collected from pigs from 664 representative smallhold farms across nine provinces in the Philippines. Isolates were identified and characterized using PCR assays. The study revealed an isolation rate of 15.8% (105/664, 95% CI: 13.0–18.6) among the sampled farms. Two hundred sixty-nine (269) S. suis isolates were recovered from 119 unique samples. Serotype 31 was the most prevalent (50/269, 95% CI: 13.9–23.2) among the other serotypes identified: 5, 6, 8, 9, 10, 11, 15, 16, 17, 21, 27, 28, and 29. The detection of the three ‘classical’ S. suis virulence-associated genes showed that 90.7% (244/269, 95% CI: 87.2–94.2) were mrp - /epf - /sly -. Multilocus sequence typing (MLST) analysis further revealed 70 novel sequence types (STs). Notably, several local isolates belonging to these novel STs formed clonal complexes (CC) with S. suis strains recovered from Spain and USA, which are major pork-exporting countries to the Philippines. This study functionally marks the national baseline knowledge of S. suis in Philippines.

The global emergence of extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae presents a significant public health threat and complicates antibiotic treatment for infections. This study aimed to determine the prevalence of ESBL-producing K. pneumoniae in a clinical setting, analyze their antimicrobial susceptibility profiles, and characterize both phenotypic and genetic determinants. A total of 507 non-duplicate clinical isolates of Enterobacterales were collected between 2019 and 2020, and third-generation cephalosporin resistance was screened by disk diffusion. Identification of K. pneumoniae was confirmed using biochemical tests and PCR with species-specific primers. Antimicrobial susceptibility testing was conducted using disk diffusion, and phenotypic ESBL production was confirmed using the combined disk method. Multiplex PCR detected ESBL genes (blaTEM, blaSHV, and blaCTX-M) and identified blaCTX-M groups. The genetic relatedness of ESBL-producing strains was assessed using the ERIC-PCR approach. Fitty-four isolates were confirmed as ESBL producers, all classified as multidrug-resistant (MDR). All ESBL-producing K. pneumoniae isolates exhibited resistance to ampicillin and cefotaxime, with high resistance rates for ciprofloxacin (98.2%), azithromycin (94.4%), piperacillin–tazobactam (88.9%), and trimethoprim (83.3%). Genotypic analysis revealed blaCTX-M was present in 94.4% of isolates, blaSHV in 87%, and blaTEM in 55.5%. The blaCTX-M-1 group was the most prevalent, accounting for 96.1% of isolates. Co-harboring of blaCTX-M, blaSHV, and blaTEM occurred in 42.6% of isolates, with co-carrying of blaCTX-M, and blaSHV was observed in 23/54 isolates. The ERIC-PCR analysis revealed 15 distinct types, indicating high genetic diversity. These findings highlight the urgent need for ongoing monitoring to control the spread of ESBL among K. pneumoniae and emphasize the importance of early detection and appropriate antibiotic selection for effectively treating infection caused by these pathogens.

Streptococcus suis is a bacterial gram-positive pathogen that causes invasive infections in swine and is also a zoonotic disease agent. Traditional molecular typing techniques such as ribotyping, multilocus sequence typing, pulse-field gel electrophoresis, or randomly amplified polymorphic DNA have been used to investigate S. suis population structure, evolution, and genetic relationships and support epidemiological and virulence investigations. However, these traditional typing techniques do not fully reveal the genetically heterogeneous nature of S. suis strains. The high-resolution provided by whole-genome sequencing (WGS), which is now more affordable and more commonly available in research and clinical settings, has unlocked the exploration of S. suis genetics at full resolution, permitting the determination of population structure, genetic diversity, identification of virulent clades, genetic markers, and other bacterial features of interest. This approach will likely become the new gold standard for S. suis strain typing as WGS instruments become more widely available and traditional typing techniques are gradually replaced.

Background: Streptococcus pneumoniae carriage is a prerequisite for clinical infections and is used to make public health decisions on vaccine licensure. Pneumococcal carriage data among high-risk Thai adults are needed before national vaccine program introduction. The association between coronavirus disease 2019 (COVID-19) and pneumococcal carriage were also investigated. Methods: During the COVID-19 pandemic, a multi-center cross-sectional study was conducted among high-risk Thai adults from September 2021 to November 2022. Pneumococcal carriage and serotypes were investigated using both conventional and molecular methods. Demographics and co-morbidities were determined for carriage while accounting for case clustering from various study sites. Results: A total of 370 individuals were enrolled. The prevalence of pneumococcal carriage, as determined by the molecular method, was 30.8 % (95 % confidence interval (CI): 26.1–35.8), while after excluding non-typeable pneumococci from the oropharyngeal sample, the carriage prevalence was 20.8 % (95 % CI: 16.79–25.31). The serotype coverage rates by pneumococcal vaccine were 12.3 %, 13.1 %, and 16.4 % for PCV13, PCV15 or PCV20, and PPSV23, respectively, while the non-vaccine type was the majority (45.1 %). The most common serotype was 19B/C (35.5 %), followed by 6 A/B/C/D (10.7 %). The age group under 65 years was associated with a higher pneumococcal carriage rate than the age group 85 and older (odds ratio (OR): 5.01, 95 % CI: 1.75–14.36). There was no significant difference between SARS-CoV-2 and carriage status. Conclusions: The prevalence of pneumococcal carriage in Thais was high. The majority of serotypes were not covered by the vaccine. Further studies on the link between carriage serotypes and disease are required. The magnitude and serotype distribution of carriage were comparable in the SARS-CoV-2 positive and negative groups.

Colistin is widely used for the prophylaxis and treatment of infectious disease in humans and livestock. However, the global food chain may actively promote the dissemination of colistin-resistant bacteria in the world. Mobile colistin-resistant (mcr) genes have spread globally, in both communities and hospitals. This study sought to genomically characterize mcr-mediated colistin resistance in 16 Escherichia coli strains isolated from retail meat samples using whole genome sequencing with short-read and long-read platforms. To assess colistin resistance and the transferability of mcr genes, antimicrobial susceptibility testing and conjugation experiments were conducted. Among the 16 isolates, 11 contained mcr-1, whereas three carried mcr-3 and two contained mcr-1 and mcr-3. All isolates had minimum inhibitory concentration (MIC) for colistin in the range 1–64 μg/mL. Notably, 15 out of the 16 isolates demonstrated successful transfer of mcr genes via conjugation, indicative of their presence on plasmids. In contrast, the KK3 strain did not exhibit such transferability. Replicon types of mcr-1-containing plasmids included IncI2 and IncX4, while IncFIB, IncFII, and IncP1 contained mcr-3. Another single strain carried mcr-1.1 on IncX4 and mcr-3.5 on IncP1. Notably, one isolate contained mcr-1.1 located on a chromosome and carrying mcr-3.1 on the IncFIB plasmid. The chromosomal location of the mcr gene may ensure a steady spread of resistance in the absence of selective pressure. Retail meat products may act as critical reservoirs of plasmid-mediated colistin resistance that has been transmitted to humans.

Extraintestinal pathogenic Escherichia coli (ExPEC), especially uropathogenic E. coli (UPEC) are responsible for urinary tract infections (UTIs), while diarrheagenic E. coli (DEC) cause foodborne illnesses. These pathogenic E. coli are a serious threat to human health and a public concern worldwide. However, the evidence on pork E. coli (PEC) harboring UPEC virulence-associated genes is currently limited. Therefore, this study aimed to determine the phylogroups, virulence genes, and their association between PEC and UPEC from UTI patients. In this study, 330 E. coli were obtained from archived stock culture isolated from pork (PEC; n = 165) and urine of patients with UTIs (UPEC; n = 165) during 2014-2022. Phylogroups, UPEC- and diarrheagenic E. coli (DEC) associated virulence genes were assessed using PCR assays. The results showed that phylogroups A (50.3%), and B1 (32.1%) were commonly found among PEC whereas phylogroups B2 (41.8%), and C (25.5%) were commonly detected in the UPEC. PEC and UPEC carried similar virulence-associated genes with different percentages. The most frequent UPEC virulence-associated gene among UPEC, and PEC strains was fimH, (93.3%, and 92.1%), followed by iucC (55.2%, and 12.7%), papC (21.8%, and 4.2%), afaC (22.4%, and 0%), hlyCA (17%, and 0.6%), cnf (16.4%, and 0.6%), and sfa/focDE (8.5%, and 4.8%). Additionally, 6 of 27 UPEC virulence-associated gene patterns were found in both PEC and UPEC strains regardless of phylogroups. Furthermore, the DEC virulence-associated genes were found in only 3 strains, one from PEC harboring eae, and two from UPEC carried fimH-bfpA or afaC-CVD432 indicating hybrid strains. Cluster analysis showed a relationship between PEC and UPEC strains and demonstrated that PEC harboring UPEC virulence-associated genes in pork may be associated with UPEC in humans. Food safety and hygiene practices during pork production chain are important procedures for minimizing cross-contamination of these strains that could be transmitted to the consumers.

Vancomycin-resistant Enterococci (VREs) have increasingly become a major nosocomial pathogen worldwide, earning high-priority category from the World Health Organization (WHO) due to their antibiotic resistance. Among VREs, vancomycin-resistant Enterococcus faecium (VREfm) is particularly concerning, frequently isolated and resistant to many antibiotics used in hospital-acquired infections. This study investigated VREfm isolates from rural tertiary hospitals in Northeastern Thailand based both antibiotic susceptibility testing and whole-genome sequencing. All isolates showed resistance to vancomycin, ampicillin, erythromycin, tetracycline, ciprofloxacin, norfloxacin, and rifampin. Nitrofurantoin and tigecycline resistance were also observed in nearly all isolates. Conversely, all isolates remained susceptible to chloramphenicol, daptomycin, and linezolid. Genomic characterization revealed that all VREfm isolates belonged to clonal complex 17 (CC17), primarily consisting of sequence type (ST) 80, followed by ST17, ST761, and ST117. Additionally, all isolates harbored numerous antimicrobial-resistant genes, including vanA, tet(L), tet(M), aac(6′)-li, ant(6)-Ia, aph(3′)-III, aac(6′)-aph(2″), aph(2″)-la, ant(9)-la, erm(B), msr(C), erm(T), erm(A), fosB, dfrG, and cfr(B). Notably, all isolates contained virulence genes, for collagen adhesin (acm) and cell wall adhesin (efafm), while hylEfm (glycosyl hydrolase) was detected in VREfm ST80. This study provided important information for understanding the genomic features of VREfm isolated from urine.

Abstract Background Streptococcus suis is a zoonotic pathogen that causes substantial economic losses in the pig industry and contributes to human infections worldwide, especially in Southeast Asia. Recently, a multiplex polymerase chain reaction (PCR) process was developed to distinguish disease-associated and non-disease-associated pathotypes of S. suis European strains. Herein, we evaluated the ability of this multiplex PCR approach to distinguish pathotypes of S. suis in Thailand. Results This study was conducted on 278 human S. suis isolates and 173 clinically healthy pig S. suis isolates. PCR identified 99.3% of disease-associated strains in the human isolates and 11.6% of non-disease-associated strains in the clinically healthy pig isolates. Of the clinically healthy pig S. suis isolates, 71.1% were classified as disease-associated. We also detected undetermined pathotype forms in humans (0.7%) and pigs (17.3%). The PCR assay classified the disease-associated isolates into four types. Statistical analysis revealed that human S. suis clonal complex (CC) 1 isolates were significantly associated with the disease-associated type I, whereas CC104 and CC25 were significantly associated with the disease-associated type IV. Conclusion Multiplex PCR cannot differentiate non-disease-associated from disease-associated isolates in Thai clinically healthy pig S. suis strains, although the method works well for human S. suis strains. This assay should be applied to pig S. suis strains with caution. It is highly important that multiplex PCR be validated using more diverse S. suis strains from different geographic areas and origins of isolation.

Abstract Streptococcus suis is a zoonotic pathogen that causes invasive infections in humans and pigs. Although S. suis serotype 2 strains are most prevalent worldwide, other serotypes are also occasionally detected. Herein, we investigated the genomes of two S. suis serotype 1 strains belonging to the clonal complex 1, which were recovered from a human patient and an asymptomatic pig, respectively. The genomes differed in pathotype, virulence-associated gene (VAG) profile, minimum core genome (MCG) typing, and antimicrobial resistance gene content. The porcine serotype 1 strain was sequence type (ST) 237 and MCG1, whereas the human serotype 1 strain was ST105 and MCG ungroupable. Both strains were susceptible to several antibiotics consisting of β-lactams, fluoroquinolones, and chloramphenicol. Resistance to tetracycline, macrolides, and clindamycin was observed, which was attributed to the genes tet(O) and erm(B). Analysis of 99 VAG revealed Hhly3, NisK, NisR, salK/salR, srtG, virB4, and virD4 were absent in both serotype 1. However, the porcine strain lacked sadP (Streptococcal adhesin P), whereas the human strain harbored sadP1. Phylogenetic analysis revealed that human S. suis ST105 strains from Vietnam were genetically the closest to the human serotype 1 strain, whereas porcine S. suis ST11 strains from China and Thailand were genetically the closest to the porcine strain.

Background Escherichia coli is the most common cause of urinary tract infections and has fluoroquinolone (FQ)-resistant strains, which are a worldwide concern. Objectives To characterize FQ-resistant determinants among 103 carbapenem-resistant E. coli (CREc) urinary isolates using WGS. Methods Antimicrobial susceptibility, biofilm formation, and short-read sequencing were applied to these isolates. Complete genome sequencing of five CREcs was conducted using short- and long-read platforms. Results ST410 (50.49%) was the predominant ST, followed by ST405 (12.62%) and ST361 (11.65%). Clermont phylogroup C (54.37%) was the most frequent. The genes NDM-5 (74.76%) and CTX-M-15 (71.84%) were the most identified. Most CREcs were resistant to ciprofloxacin (97.09%) and levofloxacin (94.17%), whereas their resistance rate to nitrofurantoin was 33.98%. Frequently, the gene aac(6′)-Ib (57.28%) was found and the coexistence of aac(6′)-Ib and blaCTX-M-15 was the most widely predominant. All isolates carried the gyrA mutants of S83L and D87N. In 12.62% of the isolates, the coexistence was detected of gyrA, gyrB, parC, and parE mutations. Furthermore, the five urinary CREc-complete genomes revealed that blaNDM-5 or blaNDM-3 were located on two plasmid Inc types, comprising IncFI (60%, 3/5) and IncFI/IncQ (40%, 2/5). In addition, both plasmid types carried other resistance genes, such as blaOXA-1, blaCTX-M-15, blaTEM-1B, and aac(6′)-Ib. Notably, the IncFI plasmid in one isolate carried three copies of the blaNDM-5 gene. Conclusions This study showed FQ-resistant determinants in urinary CREc isolates that could be a warning sign to adopt efficient strategies or new control policies to prevent further spread and to help in monitoring this microorganism.

Abstract The emergence of the mobile colistin-resistance genes mcr-1 has attracted significant attention worldwide. This study aimed to investigate the genetic features of mcr-1-carrying plasmid among carbapenem-resistant Enterobacterales (CRE) isolates and the potential genetic basis governing transmission. Seventeen mcr-harboring isolates were analyzed based on whole genome sequencing using short-read and long-read platforms. All the mcr-1-carrying isolates could be conjugatively transferred into a recipient Escherichia coli UB1637. Among these 17 isolates, mcr-1 was located on diverse plasmid Inc types, consisting of IncX4 (11/17; 64.7%), IncI2 (4/17; 23.53%), and IncHI/IncN (2/17; 11.76%). Each of these exhibited remarkable similarity in the backbone set that is responsible for plasmid replication, maintenance, and transfer, with differences being in the upstream and downstream regions containing mcr-1. The IncHI/IncN type also carried other resistance genes (bla TEM-1B or bla TEM-135). The mcr-1-harboring IncX4 plasmids were carried in E. coli ST410 (7/11; 63.6%) and ST10 (1/11; 9.1%) and Klebsiella pneumoniae ST15 (1/11; 9.1%), ST336 (1/11; 9.1%), and ST340 (1/11; 9.1%). The IncI2-type plasmid was harbored in E. coli ST3052 (1/4; 25%) and ST1287 (1/4; 25%) and in K. pneumoniae ST336 (2/4; 50%), whereas IncHI/IncN were carried in E. coli ST6721 (1/2; 50%) and new ST (1/2; 50%). The diverse promiscuous plasmids may facilitate the spread of mcr-1 among commensal E. coli or K. pneumoniae strains in patients. These results can provide information for a surveillance system and infection control for dynamic tracing.

Background and Aim: Over recent years, antimicrobial-resistant Klebsiella species in humans, animals, food animals, food products, and agricultural environments have been the center of attention due to its role in the evolution of antimicrobial resistance. The emergence of resistance to fluoroquinolones and cephalosporins of third and higher generations in Klebsiella oxytoca has not received much attention in animal husbandry compared to that in Klebsiella pneumoniae. Reports on K. oxytoca are limited in the study area. Therefore, we investigated the antimicrobial susceptibility and resistance genes in K. oxytoca isolated from slaughtered pigs in Thailand. Materials and Methods: Microbiological examination was conducted on 384 Klebsiella spp. isolates recovered from slaughtered pigs in ten provinces of Thailand. Seventy-two K. oxytoca isolates (18.75%) were examined for antimicrobial-resistant genes (β-lactamase [blaTEM, blaCTX-M, and blaSHV]) and fluoroquinolone-resistant genes (qnrA, qnrB, qnrC, qnrD, qnrS, oqxAB, aac(6')-Ib-cr, and qepA). Results: The most common genotype was blaCTX-M (58/72, 80.55%), followed by blaTEM with blaCTX-M (7/72, 9.72%) and blaTEM (6/72, 8.33%). The most common blaCTX-M group was blaCTX-M-1 (19/58, 32.76%), followed by blaCTX-M-9 (1/58, 1.72%). Plasmid-mediated quinolone resistance genes were identified in 13 (18.05%) isolates: qnrS (16.70%) and qnrB (1.4%). All 13 isolates had qnrS transferable to an Escherichia coli recipient, whereas qnrB was not detected in any transconjugants. Either blaCTX-M or blaTEM harbored by one K. oxytoca strain was transferable to an E. coli recipient. Analysis of antimicrobial susceptibility revealed that more than 90% of the blaCTX-M-carrying K. oxytoca isolates were susceptible to chloramphenicol, trimethoprim, ceftazidime, cefepime, cefotaxime, amoxicillin-clavulanic acid, piperacillin–tazobactam, and fosfomycin. All K. oxytoca isolates (13) harboring qnr were susceptible to carbapenem and ceftriaxone; however, 43 (74.13%) of the K. oxytoca isolates harboring blaCTX-M exhibited extended-spectrum β-lactamase activity. Most of the K. oxytoca isolates from pigs were highly resistant to ampicillin, azithromycin, and gentamicin. Conclusion: To prevent further transmission of Klebsiella spp. between food animals and humans, strict control of antibiotic use in clinical and livestock settings is necessary along with routine disinfection of the livestock environment and efforts to increase awareness of antimicrobial resistance transmission.

Streptococcus suis (S. suis) is an important bacterial pathogen, that causes serious infections in humans and pigs. Although numerous virulence factors have been proposed, their particular role in pathogenesis is still inconclusive. The current study explored putative peptides responsible for the virulence of S. suis serotype 2 (SS2). Thus, the peptidome of highly virulent SS2, less prevalent SS14, and rarely reported serotypes SS18 and SS19 were comparatively analyzed using a high-performance liquid chromatography-mass spectrometry method (LC-MS/MS). Six serotype-specific peptides, 2,3,4,5-tetrahydropyridine-2,6-dicarboxylate N-acetyltransferase (DapH), alanine racemase (Alr), CCA-adding enzyme (CCA), peptide chain release factor 3 (RF3), ATP synthase subunit delta (F0F1-ATPases) and aspartate carbamoyltransferase (ATCase), were expressed moderately to highly only in the SS2 peptidome with p-values of less than 0.05. Some of these proteins are responsible for bacterial cellular stability; especially, Alr was highly expressed in the SS2 peptidome and is associated with peptidoglycan biosynthesis and bacterial cell wall formation. This study indicated that these serotype-specific peptides, which were significantly expressed by virulent SS2, could serve as putative virulence factors to promote its competitiveness with other coexistences in a particular condition. Further in vivo studies of these peptides should be performed to confirm the virulence roles of these identified peptides.

Bacteriophage, a predator virus of bacteria, is an abundant biological entity in the biosphere. With ultimate applications in medicine and biotechnology, new phages are extensively being isolated and characterized. The objective of the present study was to characterize lytic bacteriophage vB_Pae-PA14 infecting Pseudomonas aeruginosa ATCC 27853 that was isolated from seawater in Thailand. vB_Pae-PA14 was subjected to whole genome phylogenetic analysis, host range test, biofilm test and characterization. Results showed that the phage belonged to a group of N4-like viruses, could infect P. aeruginosa isolates including carbapenem-resistant P. aeruginosa. The burst size of vB_Pae-PA14 was 86 plaque-forming unit/infected cells. Also, the phage showed a greater ability to control planktonic P. aeruginosa cells than the biofilm cells. Phage could withstand physical stresses especially the high salt concentration. In brief, lytic bacteriophage vB_Pae-PA14 infecting P. aeruginosa was isolated and characterized, which might be useful in further bacteriophage lytic applications.

Abstract Streptococcus suis has been well-recognized as a zoonotic pathogen worldwide, and the diversity and unpredictable adaptive potential of sporadic human strains represent a great risk to the public health. In this study, S. suis LSM178, isolated from a patient in contact with pigs and raw pork, was assessed as a hyper-virulent strain and interpreted for the virulence based on its genetic information. The strain was more invasive for Caco-2 cells than two other S. suis strains, SC19 and P1/7. Sequence analysis designated LSM178 with serotype 2 and a novel sequence type 1005. Phylogenetic analysis showed that LSM178 clustered with highly virulent strains including all human strains and epidemic strains. Compared with other strains, these S. suis have the most and the same virulent factors and a type I-89 K pathogenicity island. Further, groups of genes were identified to distinguish these highly virulent strains from other generally virulent strains, emphasizing the key roles of genes modeling transcription, cell barrier, replication, recombination and repair on virulence regulation. Additionally, LSM178 contains a novel prophage conducive potentially to pathogenicity.

Background Streptococcus suis is a zoonotic pathogen that can cause invasive infections in humans who are in close contact with infected pigs or contaminated pork-derived products. S. suis serotype 2 sequence type (ST) 1 strains are mostly associated with meningitis, whereas ST104 strains are mostly recovered from sepsis cases in humans. No data are available for comparison of the ST1 and ST104 strains at the genomic level, particularly concerning virulence-associated genes. Thus, genomic comparison of both STs was performed in this study. Methods An ST1 isolate (ID26154) from the cerebrospinal fluid of a patient with meningitis and an ST104 isolate (ID24525) from the blood of a patient with sepsis were subjected to shotgun pyrosequencing using the 454 GS Junior System. Genomic comparison was conducted between the ST1 isolate and the ST104 isolate using the Artemis Comparison Tool (ACT) to identify the region of differences (RDs) between ST1 and ST104. Results Fifty-eight RDs were unique to the ST104 genome and were mainly involved in metabolism and cell functional activities, cell wall anchored proteins, bacteriophages and mobile genetic elements, ABC-type transporters, two-component signal transductions, and lantibiotic proteins. Some virulence genes mostly found in ST1 strains were also present in the ST104 genome. Whole-genome comparison is a powerful tool for identifying genomic region differences between different STs of S. suis serotype 2, leading to the identification of the molecular basis of virulence involved in the pathogenesis of the infection.

Antimicrobial resistance (AMR) has emerged as an urgent global public health issue that requires immediate attention. Methicillin-resistant staphylococci (MRS) is a major problem, as it may cause serious human and animal infections, eventually resulting in death. This study determined the proportional distribution, genetic characteristics, and antimicrobial susceptibility of mecA- or mecC-carrying staphylococci isolated from food chain products. A total of 230 samples were taken from meat, food, fermented food, and food containers. Overall, 13.9% (32/230) of the samples were identified to have Staphylococcus aureus isolates; of those, 3.9% (9/230) were MRS, with eight mecA-positive and one mecC-positive samples, and 1.3% (3/230) methicillin-resistant Staphylococcus aureus (MRSA). MRSA strains belonging to three sequence types (ST9, ST22, and a newly identified ST), three different spa types (T005, t526, and a newly identified type), and three different SCCmec types (IV, V, and an unidentified SCCmec) were detected. Additionally, eight mecA-positive staphylococcal isolates were identified as S. haemolyticus, S. sciuri, S. simulans, and S. warneri, while the mecC-harboring isolate was S. xylosus. The enterotoxin gene, SEm, was detected at 1.56% in S. aureus, whereas SEq was detected at 0.31%, and SEi was also found in MRSA. Our study emphasizes the importance of enhanced hygiene standards in reducing the risk of occupational and foodborne MRSA infections associated with the handling or consumption of meat, food, and preserved food products.

Background and Aim: Bacterial contamination of drinking water is a leading cause of gastrointestinal infections. Cats may be at risk of water contamination from feces due to poor sanitation and hygiene. The objectives of the present study were to (1) evaluate the prevalence of coliform bacteria in cat drinking water and (2) identify possible risk factors leading to contamination. Materials and Methods: Fifty-five drinking water samples were collected from water containers used by cats (median age [range]: 5 years [8 months-15 years]) at their home. Using a sterile syringe, 50 mL water was collected directly from water containers. The water samples were stored in coliform enhancement media for 24 h and then submitted for bacterial culture. Results: The prevalence of fecal coliform contamination of cat drinking water was 67.27% (37/55; 95% confidence interval: 53.29-79.32%). There was no significant difference in the prevalence of coliform bacterial contamination of drinking water by age or gender of the cat or by water container type. However, bacterial contamination differed significantly between shorthaired cats and longhaired cats when comparing Escherichia coli (9/44 [20.45%] vs. 8/11 [72.72%], p

Methicillin-resistant staphylococci (MRS), including methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant coagulase-negative staphylococci (MRCoNS), have a global impact as a public health threat contributing significantly to morbidity, mortality, and socio-economic costs. Accurate and rapid detection of MRS results in effective antimicrobial therapy, immediate patient isolation, dissemination control, and appropriate disinfection measures. Herein, we developed an in-house cefoxitin screening broth and compared it to the cefoxitin disk diffusion method and polymerase chain reaction (PCR) for the detection of MRS. Verification of this screening broth on 52 MRSA, 37 MRCoNS, 44 methicillin-susceptible S. aureus (MSSA), and 11 MSCoNS revealed greater validity for MRSA/MSSA than for MRCoNS/MSCoNS. The kappa coefficient of 0.87 was superior for determination of MRSA and MSSA, whereas it was 0.54, which was considered poor, for determination of MRCoNS and MSCoNS. Application of this assay to screen MRSA should be useful in clinical laboratories and hospital infection-control units.

Streptococcus suis is a zoonotic pathogen that causes invasive infections in humans and pigs. Although S. suis serotype 2 is prevalent among patient and swine infections, other serotypes are occasionally detected in humans. Of these, serotype 24 clonal complex (CC) 221/234 are recognized as emerging clones of human infection. Genomic exploration of three S. suis serotype 24 CC221/234 strains revealed antimicrobial resistance genes, pathotyping, virulence-associated gene (VAG) profiles, minimum core genome (MCG) typing, and comparison of the genomes. Based on these analyzes, all three serotype 24 strains were MCG7-3 and should be classified in the intermediate/weakly virulent (I/WV) group. All selected serotype 24 strains were susceptible to several antibiotics including β-lactam, fluoroquinolone, and chloramphenicol. Resistance to tetracycline, macrolide, and clindamycin was observed and attributed to the genes tet(O) and erm(B). Genomic comparison revealed the strains S12X, LSS66, LS0L, LS0E, 92–4,172, and IMT40201 that had phylogenetic affinity with serotype 24 CC221/234. Analysis of 80 virulence-associated genes (VAG) showed that all three serotype 24 strains lacked 24 genes consisting of adhesin P, epf, hyl, ihk, irr, mrp, nadR, neuB, NisK/R, ofs, permease (SSU0835), rgg, revS, salK/R, sao, sly, spyM3_0908, srtBCD, srtF, srtG, SSU05_0473, virA, virB4, and virD4. Eleven specific sequences were identified in the 3 serotype 24 genomes that differed from the genomes of the representative strains of epidemic (E; SC84), highly virulent (HV; P1/7), I/WV (89–1,591), and avirulent (T15 and 05HAS68).

Antimicrobial resistance (AMR) is considered a serious problem in many countries, including Thailand. AMR and antibiotic resistance genes (ARGs) could transfer between humans, animals, and the environment causing a threat to human health. This study described the antibiotic resistance of Escherichia coli (E. coli) from surface water, wastewater, and discharge water in the Namsuay watershed in upper northeast Thailand. The water samples were collected in the dry and wet seasons. The 113 E. coli isolates were confirmed using a polymerase chain reaction and examined for their antibiotic susceptibility, ARGs, and genetic relationship. The results indicated that E. coli was resistant to the following classes of antibiotics: fluoroquinolone, third-generation cephalosporin, polymyxin, and carbapenem. The isolates carried the mcr-1, mcr-8, mcr-9, blaoxa-48-like, aac(6′)-bl-cr, qepA, and oqxAB genes. Phylogroup B1 was a predominant group among the E. coli in the study. In addition, the E. coli isolates from the discharge water (a hospital and a fish farm) had a higher prevalence of antibiotic resistance and harboured more ARGs than the other water sample sources. The presence of antibiotic-resistant E. coli and ARG contamination in the natural water source reflected an AMR management issue that could drive strategic policy regarding the active surveillance and prevention of AMR contamination.

Carbapenem-resistant Enterobacterales (CRE) species are top priority pathogens according to the World Health Organization. Rapid detection is necessary and useful for their surveillance and control globally. This study developed a multiplex polymerase chain reaction (mPCR) detection of the common carbapenemase genes NDM, KPC, and OXA-48-like, together with identification of Escherichia coli, and distinguished a Klebsiella pneumoniae complex to be K. pneumoniae, K. quasipneumoniae, and K. variicola. Of 840 target Enterobacterales species, 190 E. coli, 598 K. pneumoniae, 28 K. quasipneumoniae, and 23 K. variicola. with and without NDM, KPC, or OXA-48-like were correctly detected for their species and carbapenemase genes. In contrast, for the Enterobacterales species other than E. coli or K. pneumoniae complex with carbapenemase genes, the mPCR assay could detect only NDM, KPC, or OXA-48-like. This PCR method should be useful in clinical microbiology laboratories requiring rapid detection of CRE for epidemiological investigation and for tracking the trends of carbapenemase gene dynamics.

Streptococcus suis is a zoonotic pathogen causing substantial economic losses to the pig industry, as well as being a human health burden due to infections worldwide, especially in Southeast Asia. In Thailand, there was high cumulative incidence in humans during 1987–2021, mostly in males. At least five large outbreaks have been documented after the largest outbreak in China in 2005, which was related to the consumption of raw pork or dishes containing pig’s blood. The major clinical features are sepsis or meningitis, with hearing loss a major complication of S. suis disease. Thai human S. suis isolates have shown diversity in serotypes and sequence types (STs), with serotype 2 and STs 1 and 104 being major genotypes. β-Lactam antibiotics can be used in empirical treatment for human S. suis infections; however, intermediate resistance to penicillin has been reported. Reducing S. suis incidence in Thailand requires a multidimensional approach, with combined efforts from the government and public health sectors through policy, regulations, education, and active surveillance.

Antimicrobial resistance is a major public health concern globally. The most serious antimicrobial resistance problem among pathogenic bacteria is multidrug resistance (MDR). The objectives of this study were to investigate the risk factors of MDR infections and to develop a risk assessment tool for MDR Gram-negative bacteria (MDR-GNB) infections at a community hospital in rural Thailand. The study revealed 30.77% MDR-GNB among GNB strains. The most common MDR-GNB strains were 63.02% for Escherichia coli and 11.46% for Klebsiella pneumoniae. A case–control study was applied to collect clinical data between January 2016 and December 2020. Univariate logistic regression and multivariate logistic regression were used to analyze the risk factors for MDR-GNB and a risk assessment score for each factor was determined based on its regression coefficient. The risk factors for MDR-GNB infections were as follows: the presence of Enterobacteriaceae that produce extended-spectrum beta-lactamase (ESBL) (ORAdj. 23.53, 95% CI 7.00–79.09), infections occurring within the urinary tract (ORAdj. 2.25, 95% CI 1.44–3.53), and patients with a history of steroid usage (ORAdj. 1.91, 95% CI 1.15–3.19). Based on the assigned risk scores for each associated factor, the newly developed risk assessment tool for MDR-GNB infections achieved 64.54% prediction accuracy (AUC-ROC 0.65, 95% CI 0.61–0.68), demonstrating that the tool could be used to assess bacterial infection cases in community hospitals. Its use should provide practical guidance on MDR evaluation and prevention. This study was part of an antibiotic stewardship program; the study surveyed antibiotic-resistant situations in a hospital and implemented an effective risk assessment tool using key risk factors of MDR-GNB infections.

Abstract Background Active surveillance has the potential to prevent nosocomial transmission of carbapenem-resistant Acinetobacter baumannii (CRAB). We assessed whether rapid diagnosis using clinical specimen-direct loop-mediated isothermal amplification (LAMP), a rapid molecular diagnostic assay, and subsequent intervention, could reduce CRAB nosocomial transmission in intensive care units (ICUs). Methods A before and after (quasi-experimental) study was conducted in two ICUs at the Mahidol University Faculty of Medicine Ramathibodi Hospital with 3 months of observational period followed by 9 months of interventional period. All patients were screened for CRAB using both the culture and LAMP method from rectal swab and/or bronchial aspirates (intubated patients only) upon admission, weekly thereafter, and upon discharge. During the pre-intervention period, we performed contact precautions based on culture results. In contrast, during the intervention period, we initiated contact precautions within a few hours after sample collection on the basis of LAMP results. Results A total of 1335 patients were admitted to the ICUs, of which 866 patients (pre-intervention period: 187; intervention period: 679) were eligible for this study. Incidence rate of CRAB infection decreased to 20.9 per 1000 patient-days in the intervention period from 35.2 in the pre-intervention period (P

Abstract Background The etiology of severe pneumonia is frequently not identified by routine disease surveillance in Thailand. Since 2010, the Thailand Ministry of Public Health (MOPH) and US CDC have conducted surveillance to detect known and new etiologies of severe pneumonia. Methods Surveillance for severe community-acquired pneumonia was initiated in December 2010 among 30 hospitals in 17 provinces covering all regions of Thailand. Interlinked clinical, laboratory, pathological and epidemiological components of the network were created with specialized guidelines for each to aid case investigation and notification. Severe pneumonia was defined as chest-radiograph confirmed pneumonia of unknown etiology in a patient hospitalized ≤48 h and requiring intubation with ventilator support or who died within 48 h after hospitalization; patients with underlying chronic pulmonary or neurological disease were excluded. Respiratory and pathological specimens were tested by reverse transcription polymerase chain reaction for nine viruses, including Middle East Respiratory Syndrome Coronavirus (MERS-CoV), and 14 bacteria. Cases were reported via a secure web-based system. Results Of specimens from 972 cases available for testing during December 2010 through December 2015, 589 (60.6%) had a potential etiology identified; 399 (67.8%) were from children aged

Abstract Background Bloodstream infection (BSI) surveillance is essential to characterize the public health threat of bacteremia. We summarize BSI epidemiology in rural Thailand over an eight year period. Methods Population-based surveillance captured clinically indicated blood cultures and associated antimicrobial susceptibility results performed in all 20 hospitals in Nakhon Phanom (NP) and Sa Kaeo (SK) provinces. BSIs were classified as community-onset (CO) when positive cultures were obtained ≤2 days after hospital admission and hospital-onset (HO) thereafter. Hospitalization denominator data were available for incidence estimates for 2009–2014. Results From 2007 to 2014 a total of 11,166 BSIs were identified from 134,441 blood cultures. Annual CO BSI incidence ranged between 89.2 and 123.5 cases per 100,000 persons in SK and NP until 2011. Afterwards, CO incidence remained stable in SK and increased in NP, reaching 155.7 in 2013. Increases in CO BSI incidence over time were limited to persons aged ≥50 years. Ten pathogens, in rank order, accounted for > 65% of CO BSIs in both provinces, all age-groups, and all years: Escherichia coli, Klebsiella pneumoniae, Burkholderia pseudomallei, Staphylococcus aureus, Salmonella non-typhi spp., Streptococcus pneumoniae, Acinetobacter spp., Streptococcus agalactiae, Streptococcus pyogenes, Pseudomonas aeruginosa. HO BSI incidence increased in NP from 0.58 cases per 1000 hospitalizations in 2009 to 0.91 in 2014, but were higher (ranging from 1.9 to 2.3) in SK throughout the study period. Extended-spectrum beta-lactamase production among E. coli isolates and multi-drug resistance among Acinetobacter spp. isolates was common (> 25% of isolates), especially among HO cases (> 50% of isolates), and became more common over time, while methicillin-resistance among S. aureus isolates (10%) showed no clear trend. Carbapenem-resistant Enterobacteriaceae were documented in 2011–2014. Conclusions Population-based surveillance documented CO BSI incidence estimates higher than previously reported from Thailand and the region, with temporal increases seen in older populations. The most commonly observed pathogens including resistance profiles were similar to leading pathogens and resistance profiles worldwide, thus; prevention strategies with demonstrated success elsewhere may prove effective in Thailand.

Mobile colistin-resistant genes (mcr) have become an increasing public health concern. Since the first report of mcr-1 in Thailand in 2016, perspective surveillance was conducted to explore the genomic characteristics of clinical carbapenem-resistant Enterobacterales (CRE) isolates harboring mcr in 2016–2019. Thirteen (0.28%) out of 4,516 CRE isolates were found to carry mcr genes, including 69.2% (9/13) of E. coli and 30.8% (4/13) of K. pneumoniae isolates. Individual mcr-1.1 was detected in eight E. coli (61.5%) isolates, whereas the co-occurrence of mcr-1.1 and mcr-3.5 was seen in only one E. coli isolate (7.7%). No CRE were detected carrying mcr-2, mcr-4, or mcr-5 through to mcr-9. Analysis of plasmid replicon types carrying mcr revealed that IncX4 was the most common (61.5%; 8/13), followed by IncI2 (15.4%; 2/13). The minimum inhibitory concentration values for colistin were in the range of 4–16 μg/ml for all CRE isolates harboring mcr, suggesting they have 100% colistin resistance. Clermont phylotyping of nine mcr-harboring carbapenem-resistant E. coli isolates demonstrated phylogroup C was predominant in ST410. In contrast, ST336 belonged to CC17, and the KL type 25 was predominant in carbapenem-resistant K. pneumoniae isolates. This report provides a comprehensive insight into the prevalence of mcr-carrying CRE from patients in Thailand. The information highlights the importance of strengthening official active surveillance efforts to detect, control, and prevent mcr-harboring CRE and the need for rational drug use in all sectors.

Streptococcus suis, particularly S. suis serotype 2 (SS2), is an important zoonotic pathogen causing meningitis in humans worldwide. Although the proper classification of the causative and pathogenic serotype is salutary for the clinical diagnosis, cross-reactions leading to the indistinguishability of serotypes by the current serotyping methods are significant limitations. In the present study, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis of extracted peptides was developed to improve the classification of serotype of S. suis. The peptide mass fingerprint (PMFs) database of S. suis was generated from the whole-cell peptides of 32 reference strains of S. suis isolates obtained from pigs. Thirty-two human S. suis isolates from clinical cases in Thailand were used to validate this alternative serotyping method in direct comparison to the multiplex (m)PCR approach. All reference strains, representing 32 serotypes of S. suis, exhibited their individual PMFs patterns, thus allowing differentiation from one another. Highly pathogenic SS2 and SS14 were clearly differentiated from the otherwise serologically closely related SS1/2 and SS1, respectively. The developed MALDI-TOF-MS serotyping method correctly classified the serotype in 68.8% (22/32) of the same serotype isolates generated from the PMFs database; while the validity for the clinical human isolates was 62.5% (20/32). The agreement between the MALDI-TOF-MS and mPCR serotyping was moderate with a Kappa score of 0.522, considering that mPCR could correctly serotype up to 75%. The present study demonstrated that PMFs from the developed MALDI-TOF-MS-based method could successfully discriminate the previously indistinguishable highly pathogenic SS2 and SS14 from SS1/2 and SS1, respectively. Moreover, this serotyping method distinguished pathogenic SS6, and so is an alternative approach of choice to rapidly and reliably serotype clinically pathogenic S. suis isolates.

The public health systems of Southeast Asian countries are financially challenged by a comparatively higher incidence of human S. suis infections than other geographical areas. Efforts to improve practices in production settings, including improved meat inspection regulations, prevention of the slaughtering of non-healthy pigs, and enhanced hygiene practices at processing facilities, along with improvements in the pork supply chain, all appear promising for reducing food cross-contamination with S. suis. However, opportunities for intervention at the societal level are also needed to effect changes, as population behaviors such as the consumption of raw pork, blood, and offal products are important contributors to the increased incidence of human S. suis disease in Southeast Asia. A plethora of factors are associated with the consumption of these high-risk dishes, including traditional culture and knowledge, shared beliefs, socio-economic level, and personal attitudes associated with gender and/or marital status. Education and intervention in behavioral attitudes that are sensible to cultural practices and traditions may provide additional means to reduce the burden of S. suis human disease in Southeast Asia.

Vancomycin-resistant enterococci (VRE) are a major concern as microorganisms with antimicrobial resistance and as a public health threat contributing significantly to morbidity, mortality, and socio-economic costs. Among VREs, vancomycin-resistant Enterococcus faecium (VREfm) is frequently isolated and is resistant to many antibiotics used to treat patients with hospital-acquired infection. Accurate and rapid detection of VREfm results in effective antimicrobial therapy, immediate patient isolation, dissemination control, and appropriate disinfection measures. An in-house VREfm screening broth was developed and compared to the broth microdilution method and multiplex polymerase chain reaction for the detection of 105 enterococci, including 81 VRE isolates (61 E. faecium, 5 E. faecalis, 10 E. gallinarum, and 5 E. casseliflavus). Verification of this screening broth on 61 VREfm, 20 other VRE, and 24 non-VRE revealed greater validity for VREfm detection. The accuracy of this broth was 100% in distinguishing E. faecium from other enterococcal species. Our test revealed 93.3% accuracy, 97.5% sensitivity, and 79.2% specificity compared with broth microdilution and PCR detecting van genes. The kappa statistic to test interrater reliability was 0.8, revealing substantial agreement for this screening test to the broth microdilution method. In addition, the in-house VREfm screening broth produced rapid positivity after at least 8 h of incubation. Application of this assay to screen VREfm should be useful in clinical laboratories and hospital infection control units.

Francisella is an intracellular, fastidious, Gram-negative bacterium that is difficult to identify using routine microbiological methods in the laboratory. We studied the isolation of Francisella sp. (strain IDAMR664) from the blood of a patient with anti-interferon-γ (IFN-γ) autoantibodies who presented with septicemia and cholestatic hepatitis. Analysis of the strain IDAMR664 genome sequence revealed the isolate was closely related to the strain GA01-2794 that had been isolated from a human in the USA. In addition, it was clustered with F. orientalis, a fish pathogen. The isolate contained several virulence factors and had Francisella pathogenicity island pattern no. 3.

Abstract Streptococcus suis is one of the most important bacterial pathogens in the porcine industry and also a zoonotic agent. Serotype 9 is becoming one of the most prevalent serotypes within the S. suis population in certain European countries. In the present study, serotype 9 strains isolated from a country where infection due to this serotype is endemic (Spain), were compared to those recovered from Canada, where this serotype is rarely isolated from diseased pigs. For comparison purposes, strains from Brazil and the only strain isolated from a human case, in Thailand, were also incorporated. Firstly, sequence types (STs) were obtained followed by detection of putative virulence factors. Phylogenetic trees were constructed using the non-recombinant single nucleotide polymorphisms from core genomes of tested strains. Most Spanish strains were either ST123 or ST125, whereas Canadian strains were highly heterogeneous. However, the distribution of putative virulence factors was similar in both groups of strains. The fact that ST16 strains harbored more putative virulence genes and shared greater similarity with the genome of human serotype 2 strains suggests that they present a higher zoonotic and virulence potential than those from Canada and Spain. More than 80% of the strains included in this study carried genes associated with resistance to tetracycline, lincosamides and macrolides. Serotype 9 strains may be nearly 400 years old and have evolved in parallel into 2 lineages. The rapid population expansion of dominant lineage 1 occurred within the last 40 years probably due to the rapid development of the porcine industry.

A multiplex PCR was described to simultaneously detect mcr-1 and frequently occurring carbapenem-resistant genes including blaKPC, blaNDM, blaIMP, and blaOXA-48-like in a single reaction. The PCR product sizes of these 4 carbapenem-resistant genes were 232 bp, 438 bp, 621 bp, and 798 bp for blaIMP, blaOXA-48-like, blaNDM, and blaKPC, respectively, whereas mcr-1 revealed 1126 bp of PCR product. This protocol accurately detected those resistant genes in agreement with the reference strains, 127 local carbapenem-resistant Enterobacteriaceae, 8 mcr-1 carrying Enterobacteriaceae, and 62 carbapenem-susceptible Enterobacteriaceae. This method will be useful for laboratory application and surveillance of carbapenem and/or colistin-resistant bacteria. Protocol name: mPCR for mcr-1 and carbapenem-resistant genes, Keywords: PCR, mcr-1, blaNDM, blaKPC, blaIMP, blaOXA-48-like

Objectives: To investigate the incidence, clinical characteristics and cost associated with pertussis in Thai children with persistent cough. Methods: A prospective study was conducted among children aged 0–18 years with persistent cough for ≥7 days with at least one of the following: paroxysm, inspiratory whooping, or post-tussive emesis. Nasopharyngeal swabs were obtained and tested for pertussis real time polymerase chain reaction (RT-PCR). Results: 19.6% of children (28 out of 143) had pertussis confirmed by RT-PCR, 75% of cases occurred in children who were too young to complete their primary series of vaccine. Paroxysm and post-tussive emesis were the most consistent clinical features, identified in 96% and 93% of cases, respectively, whooping was found in only 18%. Pertussis cases were more likely to have household cough contact (64% versus 30%, p

Dissemination of the mobile colistin resistance gene mcr in Enterobacterales among humans, animals, and the environment is a public health issue. We characterized mcr genes in the Klebsiella pneumoniae complex (KpnC) isolated from slaughtered pigs in Thailand. The 280 KpnCs consisted of K. pneumoniae (85%), Klebsiella quasipneumoniae (8.21%), and Klebsiella variicola (6.79%). mcr genes were detected in 6.79% (19/280) of KpnC isolates, consisting of mcr-8 (n = 9; 3.21%), mcr-7 (n = 7; 2.50%), mcr-7 + mcr-8 (n = 2; 0.71%), and mcr-1 + mcr-7 (n = 1; 0.36%). K. pneumoniae predominantly carried the mcr-7 and mcr-8 genes, while K. variicola and K. quasipneumoniae harbored mcr-7 and mcr-8, respectively. Six of the nineteen mcr-harboring KpnC isolates exhibited colistin resistance, and five had mcr-1 or mcr-8 transferable to an Escherichia coli recipient. Antimicrobial susceptibility analysis revealed that all mcr-carrying KpnC isolates were susceptible to carbapenems, cefotaxime, cefepime, amoxicillin/clavulanic acid, piperacillin/tazobactam, amikacin, and fosfomycin, and had high resistance to azithromycin. Multilocus sequence analysis demonstrated that the mcr-harboring KpnC isolates were genetically diverse. A ‘One-Health’ approach is useful to combat antimicrobial-resistant bacteria through coordinating the human, animal, and environmental sectors. Hence, continuous monitoring and surveillance of mcr-carrying KpnCs throughout the pork supply chain is crucial for ensuring public health.

Streptococcus suis is a pathogen that causes invasive infections in humans and pigs. In this study, 448 S. suis isolates recovered from human infections in Thailand were characterized with regard to their antimicrobial susceptibility and antimicrobial resistance genes, including, for non-penicillin-susceptible isolates, sequence analyses of five genes encoding penicillin-binding proteins (pbp1a, pbp1b, pbp2a, pbp2b, and pbp2x). All 448 isolates were susceptible to cefepime and ceftriaxone, whereas 99.6%, 91.7%, and 72.9% of the isolates were susceptible to levofloxacin, penicillin, and chloramphenicol, respectively. Almost all isolates were resistant to tetracycline (98.2%), clindamycin (94%), erythromycin (92.4%), and azithromycin (82.6%). Genes tet(O) and ermB were the predominant resistance genes detected among macrolide- and tetracycline-resistant isolates. A total of 37 out of 448 isolates (8.2%) showed intermediately resistance to penicillin. Most of these isolates (59.5%) belonged to serotype 2-ST233. Comparison of the predicted translated sequences of five PBP proteins of a penicillin-susceptible isolate (strain P1/7) to the respective PBP sequences of ten non-penicillin-susceptible isolates revealed multiple amino acid substitutions. Isolates of CC221/234 showed highly variable amino acid substitutions in all PBP proteins. An ST104 isolate had a higher number of amino acid substitutions in PBP2X. Isolates belonging to CC233/379 had numerous substitutions in PBP2B and PBP2X. ST25 isolates exhibited fewer amino acid substitutions than isolates of other STs in all five PBPs. The antimicrobial resistance of S. suis is increasing worldwide; therefore, restrictions on antimicrobial use, continuous control, and the surveillance of this bacterium throughout the pork supply chain are crucial for ensuring public health and must be a priority concern.

Streptococcus suis (S. suis) is a zoonotic pathogen causing severe streptococcal disease worldwide. S. suis infections in pigs and humans are frequently associated with the virulent S. suis serotype 2 (SS2). Though various virulence factors of S. suis have been proposed, most of them were not essentially accounted for in the experimental infections. In the present study, we compared the peptidomes of highly virulent SS2 and SS14 in humans, the swine causative serotypes SS7 and SS9, and the rarely reported serotypes SS25 and SS27, and they were cultured in a specified culture medium containing whole blood to simulate their natural host environment. LC-MS/MS could identify 22 unique peptides expressed in the six S. suis serotypes. Under the host-simulated environment, peptides from the ABC-type phosphate transport system (SSU05_1106) and 30S ribosomal protein S2 (rpsB) were detected in the peptidome of virulent SS2 and SS14. Therefore, we suggest that these two proteins or their derived peptides might be involved in the survival of S. suis when simulated with a blood environment.

Streptococcus suis is an emerging zoonotic bacterium causing septicemia and meningitis in humans. Due to rapid disease progression, high mortality rate, and many underdiagnosed cases by time-consuming routine identification methods, alternative diagnostic testing is essential. Among 29 broadly accepted S. suis serotypes, serotypes 2 and 14 are high prevalent; however, many PCR assays showed an inability to differentiate serotype 2 from 1/2, and 1 from 14. In this study, we developed and validated a new multiplex PCR assay that facilitates the identification of only the 29 true serotypes of S. suis and simultaneously differentiates serotypes 1, 1/2, 2, and 14 within a single reaction. Importantly, the multiplex PCR could detect S. suis directly from positive hemocultures and CSF. The results revealed high sensitivity, specificity, and 100% accuracy with almost perfect agreement (κ = 1.0) compared to culture and serotyping methods. Direct detection enables a decrease in overall diagnosis time, rapid and efficient treatment, reduced fatality rates, and proficient disease control. This multiplex PCR offers a rapid, easy, and cost-effective method that can be applied in a routine laboratory. Furthermore, it is promising for developing point-of-care testing (POCT) for S. suis detection in the future.

Enhanced surveillance of invasive pneumococcal disease (IPD) in adults was conducted during April 2013-March 2018 in 10 of 47 prefectures in Japan, and a total of 1277 IPD patients were enrolled. An emergence of IPD caused by serotype 12F was identified during May 2015-March 2018 through this surveillance. 12F isolates were composed of four related sequence types. In total, 120 patients with 12F IPD were reported during this period. To characterize the clinical features of 12F IPD, the disease characteristics of these patients were compared with those of 1157 patients with non-12F IPD. Compared with the non-12F IPD patients, a significantly lower proportion of 12F IPD patients was aged 65 years or older (55% vs. 70%), vaccinated with 23-valent pneumococcal polysaccharide (4% vs. 14%), had comorbid illness (65% vs. 77%), or were immunocompromised (19% vs. 30%; all P < 0.05). No significant difference in the proportion of case fatalities was found between the two groups. The proportions of those aged 65 years or older (53% vs. 69%) and with bacteremic pneumonia (35% vs. 69%) were significantly lower in 17 patients who died from 12F IPD than in 205 patients who died from non-12F IPD (all P < 0.05). Differences in clinical features were similarly found between 12F IPD patients and patients in low- or intermediate-level invasive potential serogroups. Our data demonstrated that serotype 12F was associated with IPD in younger adults and a lower proportion of comorbid illness, including immunocompromised conditions, in adult IPD, suggesting the high invasive potential of the serotype 12F. In addition, patients who died from 12F IPD were younger and had proportionately more bacteremia without focus. These findings may provide new insight into the pathogenesis of IPD in adults caused by 12F serotype with a high invasive potential.

Carbapenem-resistant Acinetobacter pittii (CRAP) is a causative agent of nosocomial infections. This study aimed to characterize clinical isolates of CRAP from a tertiary hospital in Northeast Thailand. Six isolates were confirmed as extensively drug-resistant Acinetobacter pittii (XDRAP). The blaNDM-1 gene was detected in three isolates, whereas blaIMP-14 and blaIMP-1 were detected in the others. Multilocus sequence typing with the Pasteur scheme revealed ST220 in two isolates, ST744 in two isolates, and ST63 and ST396 for the remaining two isolates, respectively. Genomic characterization revealed that six XDRAP genes contained antimicrobial resistance genes: ST63 (A436) and ST396 (A1) contained 10 antimicrobial resistance genes, ST220 (A984 and A864) and ST744 (A56 and A273) contained 9 and 8 antimicrobial resistance genes, respectively. The single nucleotide polymorphism (SNP) phylogenetic tree revealed that the isolates A984 and A864 were closely related to A. pittii YB-45 (ST220) from China, while A436 was related to A. pittii WCHAP100020, also from China. A273 and A56 isolates (ST744) were clustered together; these isolates were closely related to strains 2014S07-126, AP43, and WCHAP005069, which were isolated from Taiwan and China. Strict implementation of infection control based upon the framework of epidemiological analyses is essential to prevent outbreaks and contain the spread of the pathogen. Continued surveillance and close monitoring with molecular epidemiological tools are needed.

The resistance of Gram-negative bacteria to colistin, mediated by plasmid-borne mcr genes, is an emerging public health concern. The complete genome sequence (4.55 Mb) of a clinical isolate of Aeromonas veronii biovar veronii obtained from a patient with septicemia was determined using short-read and long-read platforms. This isolate (C198) was found to harbor a novel mcr-3 gene, designated mcr-3.41. Isolate C198 revealed adjacent mcr-3.41 and mcr-3-like genes. It contained one chromosome and two plasmids, both of which encoded a RepB replication protein. Other antimicrobial resistance genes, including blacphA3, blaOXA-12, tetA, rsmA, and adeF, were also present. Isolate C198 was resistant to amoxicillin–clavulanate, ampicillin–sulbactam and tetracycline, and showed intermediate resistance to trimethoprim–sulfamethoxazole. The isolate was susceptible to piperacillin–tazobactam, carbapenem, third-generation cephalosporins, fluoroquinolones, chloramphenicol, and aminoglycosides. Putative virulence genes in the C198 genome encoded type II, III, and VI secretion systems; type IV Aeromonas pili; and type I fimbria, flagella, hemagglutinin, aerolysin, and hemolysins. Multilocus sequence typing revealed a novel sequence type (ST), ST720 for C198. Phylogenetic analysis of the single nucleotide polymorphisms in C198 demonstrated that the strain was closely related to A. veronii 17ISAe. The present study provides insights into the genomic characteristics of human A. veronii isolates.