Comprehensive Diagnosis of Bacterial Infection Associated with Acute Cholecystitis Using Metagenomic Approach.
ABSTRACT: Acute cholecystitis (AC), which is strongly associated with retrograde bacterial infection, is an inflammatory disease that can be fatal if inappropriately treated. Currently, bacterial culture testing, which is basically recommended to detect the etiological agent, is a time-consuming (4-6 days), non-comprehensive approach. To rapidly detect a potential pathogen and predict its antimicrobial susceptibility, we undertook a metagenomic approach to characterize the bacterial infection associated with AC. Six patients (P1-P6) who underwent cholecystectomy for AC were enrolled in this study. Metagenome analysis demonstrated possible single or multiple bacterial infections in four patients (P1, P2, P3, and P4) with 24-h experimental procedures; in addition, the CTX-M extended-spectrum ß-lactamase (ESBL) gene was identified in two bile samples (P1 and P4). Further whole genome sequencing of Escherichia coli isolates suggested that CTX-M-27-producing ST131 and CTX-M-14-producing novel-ST were identified in P1 and P4, respectively. Metagenome analysis of feces and saliva also suggested some imbalance in the microbiota for more comprehensive assessment of patients with AC. In conclusion, metagenome analysis was useful for rapid bacterial diagnostics, including assessing potential antimicrobial susceptibility, in patients with AC.
Project description:We investigated the kinetics of 4N-acetyl-pentapeptides, Ac-P1, Ac-P2, Ac-P3, and Ac-P4, regarding inhibition of mushroom tyrosinase activity. The peptides sequences of Ac-P1, Ac-P2, Ac-P3, and Ac-P4 were Ac-RSRFK, Ac-KSRFR, Ac-KSSFR, and Ac-RSRFS, respectively. The 4N-acetyl-pentapeptides were able to reduce the oxidation of l-DOPA by tyrosinase in a dose-dependent manner. Of the 4N-acetyl-pentapeptides, only Ac-P4 exhibited lag time (80 s) at a concentration of 0.5 mg/mL. The tyrosinase inhibitory effects of Ac-P4 (IC50 0.29 mg/mL) were more effective than those of Ac-P1, Ac-P2, and Ac-P3, in which IC50s were 0.75 mg/mL, 0.78 mg/mL, and 0.81 mg/mL, respectively. Kinetic analysis demonstrated that all 4N-acetyl-pentapeptides were mixed-type tyrosinase inhibitors. Furthermore, 0.1 mg/mL of Ac-P4 exhibited significant melanogenesis inhibition on B16F10 melanoma cells and was more effective than kojic acid. The melanogenesis inhibition of Ac-P4 was dose-dependent and did not induce any cytotoxicity on B16F10 melanoma cells.
Project description:The emergence of antimicrobial resistance in Klebsiella spp., including resistance to extended-spectrum cephalosporins (ESC) and fluoroquinolones, is of great concern in both human and veterinary medicine. In this study, we investigated the prevalence of antimicrobial resistance in a total of 103 Klebsiella spp. isolates, consisting of Klebsiella pneumoniae complex (KP, n = 89) and K. oxytoca (KO, n = 14) from clinical specimens of dogs and cats in Japan. Furthermore, we characterized the resistance mechanisms, including extended-spectrum ?-lactamase (ESBL), plasmid-mediated AmpC ?-lactamase (PABL), and plasmid-mediated quinolone resistance (PMQR); and assessed genetic relatedness of ESC-resistant Klebsiella spp. strains by multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Antimicrobial susceptibility testing demonstrated that resistance rates to ampicillin, cephalothin, enrofloxacin, ciprofloxacin, trimethoprim/sulfamethoxazole, cefotaxime, gentamicin, tetracycline, chloramphenicol, amoxicillin-clavulanic acid, and cefmetazole were 98.1, 37.9, 37.9, 35.9, 35.0, 34.0, 31.1, 30.1, 28.2, 14.6, and 6.8%, respectively. Phenotypic testing detected ESBLs and/or AmpC ?-lactamases in 31 of 89 (34.8%) KP isolates, but not in KO isolates. Resistances to 5 of the 12 antimicrobials tested, as well as the three PMQRs [qnrB, qnrS, and aac(6')-Ib-cr], were detected significantly more frequently in ESBL-producing KP, than in non-ESBL-producing KP and KO. The most frequent ESBL was CTX-M-15 (n = 13), followed by CTX-M-14 (n = 7), CTX-M-55 (n = 6), SHV-2 (n = 5), CTX-M-2 (n = 2), and CTX-M-3 (n = 2). Based on the rpoB phylogeny, all ESBL-producing strains were identified as K. pneumoniae, except for one CTX-M-14-producing strain, which was identified as K. quasipneumoniae. All of AmpC ?-lactamase positive isolates (n = 6) harbored DHA-1, one of the PABLs. Based on MLST and PFGE analysis, ST15 KP clones producing CTX-M-2, CTX-M-15, CTX-M-55, and/or SHV-2, as well as KP clones of ST1844-CTX-M-55, ST655-CTX-M-14, and ST307-CTX-M-15, were detected in one or several hospitals. Surprisingly, specific clones were detected in different patients at an interval of many months. These results suggest that multidrug-resistant ESBL-producing KP were clonally disseminated among companion animals via not only direct but also indirect transmission. This is the first report on large-scale monitoring of antimicrobial-resistant Klebsiella spp. isolates from companion animals in Japan.
Project description:Bacterial antimicrobial resistance mediated by the production of extended-spectrum ?-lactamases (ESBLs) is considered a major threat for treatment of Salmonella and Shigella infections. This study aimed to investigate antibiotic resistance patterns of Salmonella and Shigella spp. and presence of CTX-M from three teaching hospitals in Iran. In the present study, 58 clinical Shigella and 91 Salmonella isolates were recovered between 2009 and 2013 from 3 teaching hospitals in Iran. After culture and antimicrobial susceptibility testing, ESBL-positive isolates were subjected to further investigations. These included polymerase chain reaction (PCR) amplification and DNA sequencing of blaCTX-M-15 encoding plasmid. In both genera, high sensitivity to gentamicin and amikacin, but high resistance to ampicillin, tetracycline, and trimethoprim-sulfamethoxazole, was found. Molecular investigation showed that 31.8% isolates of Salmonella spp. and 34.48% isolates of Shigella spp. were CTX-M positive and all of them were also positive for ISEcpI. Protein translation, comparing with reference sequences, showed that all CTX-M isolates belong to CTX-M-15. The present study suggests that the resistance of ESBLs-producing Salmonella and Shigella spp. in Iran hospitals is very serious. Therefore, strategies to minimize the spread of ESBL-producing isolates should be implemented.
Project description:Dissemination of extended-spectrum-cephalosporin (ESC)-resistant Salmonella, especially extended-spectrum-?-lactamase (ESBL)-producing Salmonella, is a concern worldwide. Here, we assessed Salmonella carriage by food workers in Japan to clarify the prevalence of ESC-resistant Salmonella harboring bla CTX-M We then characterized the genetic features, such as transposable elements, of bla CTX-M-harboring plasmids using whole-genome sequencing. A total of 145,220 stool samples were collected from food workers, including cooks and servers from several restaurants, as well as food factory workers, from January to October 2017. Isolated salmonellae were subjected to antimicrobial susceptibility testing (disk diffusion method), and whole-genome sequencing was performed for Salmonella strains harboring bla CTX-M Overall, 164 Salmonella isolates (0.113%) were recovered from 164 samples, from which we estimated that at least 0.113% (95% confidence interval [CI]: 0.096 to 0.132%) of food workers may carry Salmonella Based on this estimation, 3,473 (95% CI = 2,962 to 4,047) individuals among the 3,075,330 Japanese food workers are likely to carry Salmonella Of the 158 culturable isolates, seven showed resistance to ESCs: three isolates harbored bla CMY-2 and produced AmpC ?-lactamase, while four ESBL-producing isolates harbored bla CTX-M-14 (n?=?1, Salmonella enterica serovar Senftenberg) or bla CTX-M-15 (n?=?3, S. enterica serovar Haardt). bla CTX-M-15 was chromosomally located in the S Haardt isolates, which also contained ISEcp1, while the S Senftenberg isolate contained an IncFIA(HI1)/IncHI1A/IncHI1B(R27) hybrid plasmid carrying bla CTX-M-14 along with ISEcp1 This study indicates that food workers may be a reservoir of ESBL-producing Salmonella and associated genes. Thus, these workers may contribute to the spread of bla CTX-M via plasmids or mobile genetic elements such as ISEcp1 IMPORTANCE Antimicrobial-resistant Salmonella bacteria arise in farm environments through imprudent use of antimicrobials. Subsequently, these antimicrobial-resistant strains, such as extended-spectrum-?-lactamase (ESBL)-producing Salmonella, may be transmitted to humans via food animal-derived products. Here, we examined Salmonella carriage among food handlers in Japan. Overall, 164 of 145,220 fecal samples (0.113%) were positive for Salmonella Among the 158 tested isolates, four were identified as ESBL-producing isolates carrying ESBL determinants bla CTX-M-15 or bla CTX-M-14 In all cases, the genes coexisted with ISEcp1, regardless of whether they were located on the chromosome or on a plasmid. Our findings suggest that food workers may be a reservoir of ESBL-producing strains and could contribute to the spread of resistance genes from farm-derived Salmonella to other bacterial species present in the human gut.
Project description:The emergence of extended-spectrum ?-lactamase (ESBL) and AmpC ?-lactamase producing Escherichia coli represent a contemporary public health threat. ESBL and AmpC ?-lactamase genes translocate between chromosomes and plasmids, facilitating rapid spread throughout the environment. In this study, ESBL/AmpC producing bacteria were isolated from beef cattle farms with seldom antibiotic use. Eleven farms out of 17 tested, had ESBL/AmpC producing E. coli in animals, soil, and forage samples. Fifty-nine CTX-M or CMY-2 positive E. coli isolates were further characterized with whole-genome sequencing. The isolates commonly carried CMY-2, TEM, or CTX-M genes, and over half encoded both CTX-M and TEM genes. Using comparative genomics, antimicrobial resistance genes from 12 classes of antimicrobial were identified and confirmed by antibiotic susceptibility test, revealing multidrug resistance against multiple classes of antibiotics. Virulence factors related to adherence, invasion, iron uptake, and bacterial secretion systems were shared by all isolates; some of which were identified as enteropathogenic E. coli. Phylogenetic analyses revealed a pattern of close genetic relatedness, suggesting that ESBL/AmpC producing E. coli were transmitted among farms as well as independent evolution within farms. Our results indicate that ESBL and AmpC ?-lactamases prevail in food animal production system regardless antibiotic use and have the characteristics for zoonotic transmission.
Project description:Background & objectives:Infections caused by extended-spectrum β-lactamase (ESBL)-producing Escherichia coli carrying blaCTX-M genes have been spreading globally, but there are geographical variations in the type of blaCTX-Mgenes prevalent and there are scanty data from India. This study was conducted to determine the CTX-M type ESBLs in E. coli isolates obtained from clinical specimens from patients with extra-intestinal infections attending a tertiary care hospital in south India. Methods:ESBL-producing E. coli isolated from patients with extra-intestinal infections were subjected to PCR using CTX-M group-specific primers. From a representative isolate, full-length CTX-M-15 gene was amplified and sequenced. An internal fragment of this gene was sequenced in 10 representative isolates. Results:Of the 300 isolates of E. coli tested, 88 per cent carried CTX-M genes and blaCTX-M-15was the most dominant gene present in 90 per cent of the positive isolates. Most (91%) of the isolates positive for blaCTX-M were sensitive to meropenem. Interpretation & conclusions:Our findings showed blaCTX-M-15 to be the dominant gene. Based on the data on antimicrobial susceptibility, cefoperazone-sulbactum could be an antimicrobial of choice.
Project description:The prevalence of extended-spectrum ?-lactamase-producing Escherichia coli (ESBL-producing E. coli) has recently increased worldwide. This study aims at determining the antimicrobial susceptibility patterns of a collection of clinical E. coli urine isolates and evaluating the ESBL carriage of these isolates at phenotypic and genotypic levels. A total of 100 E. coli urine isolates were collected at a tertiary healthcare centre in Riyadh from January 2018 to March 2018. Antimicrobial susceptibility testing was carried out for all isolates. ESBL production was characterized at phenotypic and genotypic levels using double-disc synergy test and polymerase chain reaction, respectively. Detection of different ESBL variants was performed using DNA sequencing. Of 100 E. coli isolates, 67 were associated with multidrug resistance (MDR) phenotype. All isolates showed variable resistance levels to all antibiotics used here expect to imipenem, where they were all imipenem-sensitive. 33 out of 100 E. coli isolates were positive for ESBLs by phenotypic and genotypic methods. Among all ESBL-positive E. coli isolates, the CTX-M was the most prevalent ESBL type (31/33 isolates; 93.94%). CTX-M-15 variant was detected in all isolates associated with CTX-M carriage. Multiple ESBL gene carriage was detected in 15/33 isolates (45.45%), where 11 (33.33%) isolates produced two different ESBL types while 4 isolates (12.12%) associated with carrying three different ESBL types. Our study documented the high antimicrobial resistance of ESBL-producing E. coli to many front-line antibiotics currently used to treat UTI patients, and this implies the need to continuously revise the local guidelines used for optimal empirical therapy for UTI patients. It also showed the high prevalence of ESBL carriage in E. coli urine isolates, with CTX-M-15 being the most predominant CTX-M variant.
Project description:Extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae have rapidly spread worldwide and pose a serious threat for health care-associated (HA) infection. We conducted molecular detection and characterization of ESBL-related bla genes, including bla(TEM), bla(SHV), bla(CTX-M), bla(VEB), bla(OXA), bla(PER), and bla(GES), among 362 isolates of ESBL-producing E. coli (n = 235) and ESBL-producing K. pneumoniae (n = 127) collected from patients who met the definition of HA infection at two major university hospitals in Thailand from December 2004 to May 2005. The prevalence of ESBL-producing E. coli and ESBL-producing K. pneumoniae, patient demographics and the susceptibilities of these bacteria to various antimicrobial agents were described. A total of 87.3% of isolates carried several bla genes. The prevalence of bla(CTX-M) was strikingly high: 99.6% for ESBL-producing E. coli (CTX-M-14, -15, -27, -40, and -55) and 99.2% for ESBL-producing K. pneumoniae (CTX-M-3, -14, -15, -27, and -55). ISEcp1 was found in the upstream region of bla(CTX-M) in most isolates. Up to 77.0% and 71.7% of ESBL-producing E. coli and ESBL-producing K. pneumoniae, respectively, carried bla(TEM); all of them encoded TEM-1. ESBL-producing K. pneumoniae carried bla(SHV) at 87.4% (SHV-1, -2a, -11, -12, -27, -71, and -75) but only at 3.8% for ESBL-producing E. coli (SHV-11 and -12). bla genes encoding VEB-1 and OXA-10 were found in both ESBL-producing E. coli (8.5% and 8.1%, respectively) and ESBL-producing K. pneumoniae (10.2% and 11.8%, respectively). None of the isolates were positive for bla(PER) and bla(GES). Pulsed-field gel electrophoresis analysis demonstrated that there was no major clonal relationship among these ESBL producers. This is the first study to report CTX-M-3, CTX-M-27, CTX-M-40, SHV-27, SHV-71, and SHV-75 in Thailand and to show that CTX-M ESBL is highly endemic in the country.
Project description:The actual state of intestinal long-term colonization by extended-spectrum ?-lactamase (ESBL)-producing Escherichia coli in healthy Japanese people remains unclear. Therefore, a total of 4,314 fecal samples were collected from 2,563 food handlers from January 2010 to December 2011. Approximately 0.1 g of each fecal sample was inoculated onto a MacConkey agar plate containing cefotaxime (1 ?g/ml). The bacterial colonies that grew on each plate were checked for ESBL production by the double-disk synergy test, as recommended by the Clinical and Laboratory Standards Institute. The bacterial serotype, antimicrobial susceptibility, pulsotype, sequence type (ST), and ESBL genotype were checked, and the replicon types of plasmids harboring the ESBL gene were also determined after conjugation experiments. ESBL producers were recovered from 70 (3.1%) of 2,230 participants who were checked only once. On the other hand, ESBL producers were isolated at least once from 52 (15.6%) of 333 participants who were checked more than twice, and 13 of the 52 participants carried ESBL producers for from more than 3 months to up to 2 years. Fluoroquinolone (FQ)-resistant E. coli strains harboring bla(CTX-M) were repeatedly recovered from 11 of the 13 carriers of bla(CTX-M)-harboring E. coli. A genetically related FQ-resistant E. coli O25b:H4-ST131 isolate harboring bla(CTX-M)-27 was recovered from 4 of the 13 carriers for more than 6 months. Three FQ-resistant E. coli O1:H6-ST648 isolates that harbored bla(CTX-M-15) or bla(CTX-M)-14 were recovered from 3 carriers. Moreover, multiple CTX-M-14- or CTX-M-15-producing E. coli isolates with different serotypes were recovered from 2 respective carriers. These findings predict a provable further spread of ESBL producers in both community and clinical settings.
Project description:During bacterial chemotaxis, transmembrane chemoreceptor arrays regulate autophosphorylation of the dimeric histidine kinase CheA. The five domains of CheA (P1-P5) each play a specific role in coupling receptor stimulation to CheA activity. Biochemical and X-ray scattering studies of thermostable CheA from Thermotoga maritima determine that the His-containing substrate domain (P1) is sequestered by interactions that depend upon P1 of the adjacent subunit. Non-hydrolyzable ATP analogs (but not ATP or ADP) release P1 from the protein core (domains P3P4P5) and increase its mobility. Detachment of both P1 domains or removal of one within a dimer increases net autophosphorylation substantially at physiological temperature (55°C). However, nearly all activity is lost without the dimerization domain (P3). The linker length between P1 and P3 dictates intersubunit (trans) versus intrasubunit (cis) autophosphorylation, with the trans reaction requiring a minimum length of 47 residues. A new crystal structure of the most active dimerization-plus-kinase unit (P3P4) reveals trans directing interactions between the tether connecting P3 to P2-P1 and the adjacent ATP-binding (P4) domain. The orientation of P4 relative to P3 in the P3P4 structure supports a planar CheA conformation that is required by membrane array models, and it suggests that the ATP lid of CheA may be poised to interact with receptors and coupling proteins. Collectively, these data suggest that the P1 domains are restrained in the off-state as a result of cross-subunit interactions. Perturbations at the nucleotide-binding pocket increase P1 mobility and access of the substrate His to P4-bound ATP.