Pulsed-field Gel Electrophoresis Research Articles

Genetic relatedness of third-generation cephalosporin-resistant Escherichia coli among livestock, farmers, and patients in Japan

ObjectivesThe third-generation cephalosporin (3GC)-resistant E. coli strains have been detected worldwide in humans and animals. Hence, in this study, we evaluated the prevalence and genetic characteristics of 3GC-resistant E. coli in livestock, farmers, and patients to further analyse if livestock serves as a potential reservoir of antimicrobial-resistant bacteria. MethodsFaecal samples were collected from 330 healthy livestock (216 cattle and 114 swine), 61 healthy livestock farmers (52 cattle farmers and 9 swine farmers), and 68 non-duplicate 3GC-resistant E. coli isolates were also obtained from the clinical specimens of patients in Japan between 2013 and 2015. Genes associated with resistance in 3GC-resistant E. coli were identified using polymerase chain reaction (PCR) and DNA sequencing. Genotypic diversity was determined by multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). ResultsWe obtained 39 and 17 non-duplicated 3GC-resistant E. coli strains from healthy livestock (33 cattle and six swine) and livestock farmers, respectively. All isolates carried either CTX-M-type extended-spectrum β-lactamase or plasmid-mediated AmpC β-lactamase genes, with CTX-M-14 being the most frequent. CTX-M producers from livestock and patients belonged to 22 and 19 different sequence types (STs), respectively, and only three STs were the same. Among the 3GC-resistant E. coli from livestock and farmers, three types of CTX-M producers have shown similar characteristics (CTX-M genotype, ST, PFGE patterns, and antimicrobial susceptibilities) and were identified as clonal isolates shared among their farms. ConclusionsOur study findings indicate that CTX-M-14 is predominant in Japan. No distinct relationship was observed between the 3GC-resistant E. coli isolated from livestock and patients; however, some clonal relatedness was observed between the isolates from livestock and farmers due to their close contact.

Spontaneous Genomic Variation as a Survival Strategy of Nosocomial Staphylococcus haemolyticus.

Staphylococcus haemolyticus is one of the most important nosocomial human pathogens frequently isolated in bloodstream and medical device-related infections. However, its mechanisms of evolution and adaptation are still poorly explored. To characterize the strategies of genetic and phenotypic diversity in S. haemolyticus, we analyzed an invasive strain for genetic and phenotypic stability after serial passage in vitro in the absence and presence of beta-lactam antibiotics. We performed pulsed-field gel electrophoresis (PFGE) of the culture and analyzed five colonies at seven time points during stability assays for beta-lactam susceptibility, hemolysis, mannitol fermentation, and biofilm production. We compared their whole genomes and performed phylogenetic analysis based on core single-nucleotide polymorphisms (SNPs). We observed a high instability in the PFGE profiles at the different time points in the absence of antibiotic. Analysis of WGS data for individual colonies showed the occurrence of six large-scale genomic deletions within the oriC environ, smaller deletions in non-oriC environ regions, and nonsynonymous mutations in clinically relevant genes. The regions of deletion and point mutations included genes encoding amino acid and metal transporters, resistance to environmental stress and beta-lactams, virulence, mannitol fermentation, metabolic processes, and insertion sequence (IS) elements. Parallel variation was detected in clinically significant phenotypic traits such as mannitol fermentation, hemolysis, and biofilm formation. In the presence of oxacillin, PFGE profiles were overall stable over time and mainly corresponded to a single genomic variant. Our results suggest that S. haemolyticus populations are composed of subpopulations of genetic and phenotypic variants. The maintenance of subpopulations in different physiological states may be a strategy to adapt rapidly to stress situations imposed by the host, particularly in the hospital environment. IMPORTANCE The introduction of medical devices and antibiotics into clinical practice have substantially improved patient quality of life and contributed to extended life expectancy. One of its most cumbersome consequences was the emergence of medical device-associated infections caused by multidrug-resistant and opportunistic bacteria such as Staphylococcus haemolyticus. However, the reason for this bacterium's success is still elusive. We found that in the absence of environmental stresses, S. haemolyticus can spontaneously produce subpopulations of genomic and phenotypic variants with deletions/mutations in clinically relevant genes. However, when exposed to selective pressures, such as the presence of antibiotics, a single genomic variant will be recruited and become dominant. We suggest that the maintenance of these cell subpopulations in different physiological states is an extremely effective strategy to adapt to stresses imposed by the host or the infection environment and might contribute for S. haemolyticus survival and persistence in the hospital.

Molecular characterization of antibiotic resistance, aminoglycoside and PmrA genes among foodborne and clinical Acinetobacter spp.

The assessment of antibiotic resistance and related genes of foodborne Acinetobacter spp. and the analysis of whether they are genetically related to clinical infection-agent strains are crucial in terms of sustainability of food safety. The study at hand investigated antibiotic resistance, aminoglycoside-modifying enzyme (AME), and colistin resistance (PmrA) genes, clonal relationships while evaluating a possible correlation between antibiotic resistance and related genes between 27 foodborne and 50 clinical Acinetobacter spp. in Turkey. Antimicrobial susceptibilities, (AME), PmrA genes, and clonal relatedness of the strains were performed by disc diffusion, PCR, and Pulsed Field gel Electrophoresis (PFGE) methods, respectively. The aph-AI, aph-6, anth (3’’)-I, aadA1, aadB and PmrA genes were found as (24, 48%), (11, 22%), (7, 14%), (1, 2%), (2, 4%), and (46, 92%) respectively in clinical strains. This rate was found as (14, 51.9%), (16, 59.3%), (19, 70.4%), (2, 7.4%), (0, 0%), and (27, 100%) respectively in foodborne isolates. A positive correlation existed between the number of aph-AI gene positivity and trimethoprim-sulfamethoxazole and gentamycin resistance; anth (3’’)-I gene positivity, and colistin resistance; PmrA gene positivity and piperacillin-tazobactam, ceftazidime, meropenem, amikacin, and imipenem resistance in clinical strains (P

PFGE analysis of Listeria monocytogenes isolates from milk, bovine clinical and bovine environment origin from Nagpur

Listeria monocytogenes, a foodborne pathogen, causes listeriosis, that mainly affects immunocompromised persons. Epidemiological investigation and molecular subtyping can be helpful in linking the specific sources of infection. Thirty-eight isolates of Listeria monocytogenes recovered from the bovine environment, milk, and bovine clinical cases were characterized by serotyping and pulse field gel electrophoresis (PFGE) to correlate the genetic links between the isolates. Serotyping revealed 3 L. monocytogenes isolates to be of serovar group 4b, 4d and 4e, 34 isolates of serovar group 1/2b, 4b, 3b, 4d, 4e, and one isolate to be serovar group 1/2a, 1/2c,3a, and 3c. The PFGE analysis revealed 38 L. monocytogenes isolate from seven different clusters combined ApaI and AscI with 80% relative genetic similarity. In comparison majority of the strains showed location and serovar wise homology in PFGE pattern than source wise. It was also observed that at the place of collection, the organisms circulate in the environment. The present findings suggested that the dairy/bovine farm environment is a potential source of contamination of L. monocytogenes in milk and animals.

Drug Resistance Genes and Molecular Epidemiological Characteristics of Carbapenem-Resistant Klebsiella pneumonia.

The global prevalence of carbapenem-resistant Klebsiella pneumoniae (CRKP) has become a serious challenge for nosocomial infection and attracted worldwide attention. This study explored the drug resistance genes and molecular characteristics for CRKP, providing a reference for nosocomial prevention and control. A total of 42 CRKP isolates were collected from the First Affiliated Hospital of Gannan Medical University (Ganzhou, China) from January 2018 to February 2021. The drug resistance of CRKP was tested by the VitekII Compact system. Drug resistance gene expression was detected by poly-merase chain reaction (PCR), and molecular typing was performed by pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). All the 42 CRKP isolates were multi-drug resistant. Among them, 35 isolates (83.3%) produced blaKPC-2 and 12 isolates (28.6%) produced blaNDM-1. The detection rate of blaIMP-4 and blaOXA-48 was 2.4% (1/42), respectively. Twelve isolates (28.6%) carried both blaKPC-2 and blaNDM-1, one isolate (2.4%) carried both blaKPC-2 and blaIMP-4, and one isolate (2.4%) carried blaKPC-2, blaNDM-1 and blaOXA-48. A variety of other extended-spectrum beta-lactamases (ESBLs) were also detected. All 42 isolates carried blaSHV and blaCTX-M-1, 27 isolates (64.3%) carried blaTEM and 12 isolates (28.6%) carried blaCTX-M-9. The MLST data classified the 42 CRKP isolates into 11 sequence types, mainly ST11, accounting for 61.9% (26/42), of which 92.3% of isolates (24/26) carrying blaKPC-2. The PFGE results demonstrated that the 42 CRKP isolates could be divided into 20 clusters A-T, with cluster A (26.2%, 11/42) and cluster H (21.4%, 9/42) dominating, which were all ST11. The CRKP isolates were severely multi-drug resistant, and the main resistant gene was blaKPC-2 production, carrying multiple ESBLs genes simultaneously. The MLST and PFGE revealed that the ST11-blaKPC-2 Klebsiella pneumoniae was the main clonotype. Our findings may offer help to antibiotics selection and nosocomial infection prevention and control.

Bacterial and fungal microbiota of mould‐ripened cheese produced in Konya

Bacterial and fungal diversities of 24 mould‐ripened cheeses originating from Konya‐Türkiye were examined by metagenomic analysis. Firmicutes phylum, Enterococcus, Clostridium sensu stricto and Lactobacillus (Levilactobacillus) genera were the dominant bacteria. Ascomycota phylum and Penicillium and Pichia genera and Penicillium roqueforti and Pichia membranifaciens species were dominant fungi. Enterococcus faecium (n = 30) and Enterococcus faecalis (n = 6) were identified, and all strains were susceptible to penicillin, ampicillin, vancomycin, teicoplanin, chloramphenicol and linezolid. The highest resistance (n = 14) was against rifampin. Tetracycline resistance was determined in two strains. Biofilm‐forming ability was found in nine E. faecium and 1 E. faecalis. E. faecium strains revealed 40–88.9%, and E. faecalis showed 59.2–100% homology by pulsed field gel electrophoresis.

Intra-Abdominal Hypertension Contributes to the Development of Ventilator-Associated Pneumonia from Intestinal Bacteria.

Ventilator-associated pneumonia (VAP) is an ICU (intensive care unit)-acquired pulmonary parenchymal infection that is complicated by mechanical ventilation and is associated with high morbidity and mortality. Klebsiella pneumoniae (KPN) is known to asymptomatically colonize the gastrointestinal tract and may increase the incidence of corresponding VAP. Our study aims were to investigate the exact origin of the carbapenem-resistant Klebsiella pneumoniae (CRKP) causing VAP in our patient. Various environmental samples, including the patient's anal swab, were collected in order to find the source of the bacteria. Minimum inhibitory concentrations (MICs) for antimicrobial agents were determined according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI); resistant genes were detected by using PCR and sequencing; clone relationships were analyzed by using multilocus-sequence typing (MLST) and pulsed field gel electrophoresis (PFGE). The IAP values were obtained via urinary catheter. One CRKP strain was detected in the patient's anal swab; this strain was confirmed with the same gene type as the strain isolated from the sputum. We found that the patient's intra-abdominal pressure (IAP) was 29.41, 27.06, 24.12, and 22.66 mmHg; the IAP was either equal to or above 12 mmHg, on the operation day and the following three days. Intra-abdominal hypertension (IAH) occurred during the patient's hospitalization and was considered to be caused by the surgical procedure. Meanwhile, we found that there was a correlation between IAH and the detection of CRKP in the sputum. The findings suggested that his VAP was caused by intestinal colonial KPN, and not from the environment. Our research illustrated that the ST11 KPC-2-producing strain colonized the intestinal tract and caused the development of VAP when the IAP was elevated. Routine screening for the intestinal carriage of CRKP, among patients in ICUs, can limit and prevent current and future outbreaks.

Outbreak of Arcobacter butzleri? An emerging enteropathogen

BackgroundArcobacter butzleri is a gram-negative rod, with microaerobic growth at an optimal temperature of 37°C. It was reported to be the fourth most common Campylobacter-like organism isolated from patients with diarrhoea. ObjectiveCharacterise a potential outbreak of A. butzleri detected in a short period of time in the University Hospital Marqués de Valdecilla. MethodsEight strains of A. butzleri were detected in our hospital in only two months. Isolates were identified by MALDI-TOF MS system and 16S rDNA sequencing. Enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) and Pulsed Field Gel Electrophoresis (PFGE) were carried out to assess clonal relationship. Gradient strips (Etest) were used to determine susceptibility by agar diffusion. ResultsERIC-PCR and PFGE confirmed the lack of clonal relationship between strains. Erythromycin or ciprofloxacin might be appropriate for antibiotic treatment of infections. ConclusionsA. butzleri is an emerging pathogen with increasing incidence, and may be underestimated.

Selection of the methylotrophic yeast Ogataea minuta as a high-producing host for heterologous protein expression

Three Ogataea minuta var. minuta strains have been deposited as NBRC 0975, NBRC 10402, and NBRC 10746 in the National Institute of Technology and Evaluation (NITE) Biological Resource Center (NBRC) collection. We investigated the ability to produce secretory proteins and several genotypic and phenotypic characteristics in order to select the best strain for heterologous protein expression. NBRC 10746 showed the best performance as evaluated by Cypridina noctiluca luciferase expression. Subsequently, clone #5-30 named tat06213, which was obtained by single-colony isolation from NBRC 10746, was established as a promising host for heterologous protein expression. To deepen our understanding of the characteristics of O.minuta var. minuta strains, sequence analysis of the D1/D2 domain of large subunit rRNA was conducted and the resulting phylogenetic tree derived from the D1/D2 domain showed that NBRC 10402 and NBRC 10746 were grouped into a different cluster far from NBRC 0975. Furthermore, a chromosome structure topology with electrophoretic karyotype and AOX1 loci analyzed by pulsed-field gel electrophoresis with Southern blotting showed different chromosome patterns and AOX1-hybridization loci among the strains. Additionally, the sequences of the promoter regions of the cloned AOX1 genes were not identical among the three strains. These findings might explain the differences in heterologous protein expression among the tested O. minuta var. minuta strains.

Different Roles of Wild Boars and Livestock in Salmonella Transmission to Humans in Italy.

Wild boar (Sus scrofa) is the most widely distributed large wildlife mammal worldwide. To investigate the transmission of Salmonella enterica amongst wild boars (Sus scrofa), humans, and livestock, we compared via pulsed-field gel electrophoresis and whole genome sequences the isolates of S. enterica serovar Typhimurium (biphasic and monophasic variants) and Enteritidis collected from wild boars, food-producing animals, and human patients in Emilia-Romagna region (Northern Italy) between 2017 and 2020. Specifically, we analysed 2175 isolates originated from human (1832), swine (117), bovine (128), poultry (76), and wild boar (22). The genomic analyses showed that wild boars shared most of their lineages of biphasic Typhimurium with bovines and most of Enteritidis with poultry, whilst we did not find any lineage shared with swine. Moreover, almost 17% of human biphasic Typhimurium and Enteritidis belonged to genomic clusters including wild boar isolates, but the inclusion of bovine and poultry isolates in the same clusters and the peculiar spatial distribution of the isolates suggested that human cases (and wild boar infections) likely originated from bovines and poultry. Consequently, wild boars appear not to play a significant role in infecting humans with these serovars, but seem to get infected themselves from livestock, probably through the environment.

Emergence of OXA-484-Producing Klebsiella variicola in China.

The frequent and inappropriate use of antibiotics has caused a dramatic rise in the number, species, and degree of multi-drug resistant bacteria, making them more prevalent and difficult to treat. In this context, the aim of the present study was to characterize the OXA-484-producing strains isolated from a perianal swab of a patient by using whole-genome analysis. In this study, carbapenemase-producing Klebsiella variicola was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), average nucleotide identity (ANI) and PCR. S1 nuclease pulsed-field gel electrophoresis (S1-PFGE) and Southern blotting were utilized to characterize the plasmid profiles of K. variicola 4717. In particular, WGS was performed to obtain genomic information on this clinical isolate, and assemble all the plasmids of the bla OXA-484-harboring strain. The antimicrobial susceptibility pattern of K. variicola 4717 revealed that it was resistant to a range of antibiotics, including aztreonam, imipenem, meropenem, ceftriaxone, cefotaxime, ceftazidime, levofloxacin, ciprofloxacin, piperacillin-tazobactam, methylene-sulfamer oxazole, amoxicillin-clavulanic acid, cefepime, and tigecycline. Its susceptibility to chloromycin was intermediate, while it was still susceptible to amikacin, gentamicin, fosfomycin, and polymyxin B. The presence of two companion plasmids, p4717_1 and p4717_2, together with a plasmid carrying the bla OXA-484 gene was observed. An in-depth investigation of p4717-OXA-484 uncovered that it is an IncX3-type plasmid and shares a similar segment encoded by IS26. Given the similar genetic background, it was conceivable that bla OXA-484 could have developed from bla OXA-181 through a series of mutations. Herein, we described the first genome sequence of K. variicola strain harbouring the class D β-actamase bla OXA-484 in an Inc-X3-type plasmid. Our work also uncovered the genetic characterization of K. variicola 4717 and the importance of initiating antimicrobial detection promptly.

Bacterial chondronecrosis with osteomyelitis related Enterococcus cecorum isolates are genetically distinct from the commensal population and are more virulent in an embryo mortality model

Bacterial chondronecrosis with osteomyelitis (BCO) is a common cause of broiler lameness. Bacteria that are found in BCO lesions are intestinal bacteria that are proposed to have translocated through the intestinal epithelium and have spread systemically. One of the specific bacterial species frequently isolated in BCO cases is Enterococcus cecorum. In the current study, caecal isolates were obtained from birds derived from healthy flocks (12 isolates from 6 flocks), while isolates derived from caeca, colon, pericardium, caudal thoracic vertebrae, coxo-femoral joint, knee joint and intertarsal joint (hock) were obtained from broilers derived from BCO outbreaks (111 isolates from 10 flocks). Pulsed field gel electrophoresis was performed to determine similarity. Clonal E. cecorum populations were isolated from different bones/joints and pericardium from animals within the same flock, with intestinal strains carrying the same pulsotype, pointing to the intestinal origin of the systemically present bacteria. Isolates from the intestinal tract of birds from healthy flocks clustered away from the BCO strains. Isolates from the gut, bones/joints and pericardium of affected animals contained a set of genes that were absent in isolates from the gut of healthy animals, such as genes encoding for enterococcal polysaccharide antigens (epa genes), cell wall structural components and nutrient transporters. Isolates derived from the affected birds induced a significant higher mortality in the embryo mortality model as compared to the isolates from the gut of healthy birds, pointing to an increased virulence.

Heat-resistant and biofilm-forming Escherichia coli in pasteurized milk from Brazil.

Escherichia coli harboring a transmissible locus of stress tolerance (tLST) and the ability to form biofilms represent a serious risk in dairy production. Thus, we aimed to evaluate the microbiological quality of pasteurized milk from two dairy producers in Mato Grosso, Brazil, with a focus on determining the possible presence of E. coli with heat resistance (60°C/6min), biofilm-forming potential phenotypes and genotypes, and antimicrobial susceptibility. For this, fifty pasteurized milk samples from producers named A and B were obtained for 5weeks to investigate the presence of Enterobacteriaceae members, coliforms, and E. coli. For heat resistance, E. coli isolates were exposed to a water bath at 60°C for 0 and 6min. In antibiogram analysis, eight antibiotics belonging to six antimicrobial classes were analyzed. The potential to form biofilms was quantified at 570nm, and curli expression by Congo Red was analyzed. To determine the genotypic profile, we performed PCR for the tLST and rpoS genes, and pulsed-field gel electrophoresis (PFGE) was used to investigate the clonal profile of the isolates. Thus, producer A presented unsatisfactory microbiological conditions regarding Enterobacteriaceae and coliforms for weeks 4 and 5, while all samples analyzed for producer B were contaminated at above-the-limit levels established by national and international legislation. These unsatisfactory conditions enabled us to isolate 31 E. coli from both producers (7 isolates from producer A and 24 isolates from producer B). In this way, 6 E. coli isolates (5 from producer A and 1 from producer B) were highly heat resistant. However, although only 6 E. coli showed a highly heat-resistant profile, 97% (30/31) of all E. coli were tLST-positive. In contrast, all isolates were sensitive to all antimicrobials tested. In addition, moderate or weak biofilm potential was verified in 51.6% (16/31), and the expression of curli and presence of rpoS was not always related to this biofilm potential. Therefore, the results emphasize the spreading of heat-resistant E. coli with tLST in both producers and indicate the biofilm as a possible source of contamination during milk pasteurization. However, the possibility of E. coli producing biofilm and surviving pasteurization temperatures cannot be ruled out, and this should be investigated.

Phenotypic and genotypic characterization of Listeria monocytogenes in clinical ruminant cases in Korea

Listeria monocytogenes, a foodborne human and veterinary pathogen, is associated with high mortality rates in ruminants. However, no studies have investigated the antimicrobial resistance of L. monocytogenes isolates from clinical ruminant cases. This study aimed to determine the phenotypic and genotypic characteristics of L. monocytogenes isolates from clinical cases of Korean ruminants. We collected 24 L. monocytogenes isolates from aborted bovine fetuses and goats presenting with listeriosis-related symptoms. The isolates were subjected to PCR serogrouping, conventional serotyping, virulence gene detection, and antimicrobial susceptibility testing. Furthermore, pulsed-field gel electrophoresis and multilocus sequence typing were used to classify and compare genetic diversity among the isolates, including human L. monocytogenes isolates. The most prevalent L. monocytogenes serotypes were 4b (Ⅳb), 1/2a (Ⅱa; Ⅱc), and 1/2b (Ⅱb). All isolates harbored the virulence genes; however, llsX-encoding listeriolysin were identified only in serotypes 4b and 1/2b. All isolates, including two found in humans, formed three genetically diverse pulsed-field gel electrophoresis clusters according to serotype, lineage, and sequence type. The most prevalent sequence type was ST1, followed by ST365 and ST91. The isolates from ruminants with listeriosis were resistant to oxacillin and ceftriaxone and showed diverse lineage, serotype (serogroup), and sequence type characteristics. Considering that the atypical sequence types exhibited clinical manifestations and histopathological lesions, further study is needed to elucidate the pathogenicity of genetically diverse ruminant L. monocytogenes isolates. Furthermore, continuous monitoring of antimicrobial resistance is required to prevent the emergence of L. monocytogenes strains resistant to common antimicrobials.

Molecular epidemiology and transmission of rmtB-positive Escherichia coli among ducks and environment

This study aimed to investigate the transmission and molecular epidemiological characteristics of the rmtB gene in Escherichia coli (E. coli) strains isolated from duck farms in Guangdong Province of China from 2018 to 2021. A total of 164 (19.4%, 164/844) rmtB-positive E. coli strains were recovered from feces, viscera, and environment. We performed antibiotic susceptibility tests, pulsed-field gel electrophoresis (PFGE), and conjugation experiments. We obtained the genetic context of 46 rmtB-carrying E. coli isolates and constructed a phylogenetic tree via whole genome sequencing (WGS) and bioinformatic analysis. The isolation rate of rmtB-carrying E. coli isolates in duck farms increased yearly from 2018 to 2020 but decreased in 2021. All rmtB-harboring E. coli strains were multidrug resistant (MDR), and 99.4% of the strains were resistant to more than 10 drugs. Surprisingly, duck- and environment-associated strains similarly showed high MDR. Conjugation experiments revealed that the rmtB gene horizontally cocarried blaCTX-M and blaTEM gene dissemination via IncFII plasmids. Insertion sequences IS26, ISCR1, and ISCR3 were closely associated with the spread of rmtB-harboring E. coli isolates. WGS analysis indicated that ST48 was the most prevalent sequence type. The results of single nucleotide polymorphism (SNP) differences revealed potential clonal transmission between ducks and the environment. Based on One Health principles, we need to strictly use veterinary antibiotics, monitor the distribution of MDR strains, and evaluate the impact of plasmid-mediated rmtB gene on human, animal, and environmental health.

Evaluation of IR Biotyper for carbapenem-resistant Pseudomonas aeruginosa typing and its application potential for the investigation of nosocomial infection.

Pseudomonas aeruginosa is one of the most common opportunistic pathogens causing severe nosocomial infections for its patterns of multidrug resistance, particularly for carbapenems. Timely epidemiological surveillance could greatly facilitate infection control of P. aeruginosa and many deadly pathogens alike. IR Biotyper (IRBT), is a novel real-time typing tool, based on a Fourier-transform infrared (FTIR) spectroscopy system. It is critical to comprehensively establish and evaluate the feasibility of IRBT in P. aeruginosa strain typing. In the current study, we first established standards and schemes for its routine laboratory application, and we found that Mueller-Hinton agar plates give better discriminatory power than blood agar plates. Data showed that the cut-off value of 0.15 with an additional 0.025 range was optimal. Secondly, 27 clinically isolated carbapenem-resistant P. aeruginosa (CRPA) strains collected from October 2010 to September 2011 were evaluated for typing effectiveness by comparing IRBT to the other commonly used typing methods, such as multi-locus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE) and whole-genome sequencing (WGS)-based typing. When using WGS-based typing as the reference method, the typing method of FTIR spectroscopy (AR = 0.757, SID = 0.749) could better cluster P. aeruginosa strains than MLST and in silico serotyping (AR = 0.544, SID = 0.470). Though PFGE showed the highest discriminatory power, low concordance was observed between PFGE and the other methods. Above all, this study demonstrates the utility of the IRBT as a quick, low-cost, real-time typing tool for the detection of CRPA strains.

Genotyping and antimicrobial resistance profiles of chicken originated Salmonella Enteritidis isolates.

Salmonellosis is a common foodborne zoonosis worldwide. The most common Salmonella serovar in humans is Salmonella enterica subsp. enterica serovar Enteritidis (50.3%) in the world. The main transmission route for S. Enteritidis is consumption of contaminated poultry products. Therefore, it is important to determine the diversity and spread of chicken-originated S. Enteritidis isolates in order to monitor and control salmonellosis. Pulsed-field gel electrophoresis (PFGE) and multiple locus variable number of tandem repeats analysis (MLVA) are frequently used for typing of S. Enteritidis isolates. This study aimed to determine the antimicrobial resistance (AMR) profiles and MLVA and PFGE genotypes of chicken-originated S. Enteritidis isolates. A total of 200 S. Enteritidis isolated from chicken broiler, layer, and breeder flocks from different locations in Turkey were investigated by Kirby-Bauer disk diffusion method, PFGE, and MLVA. The AMR test indicated that 57% of the S. Enteritidis isolates were susceptible to all antimicrobials, while 39% were resistant to at least one antimicrobial. The highest resistance (25%) was against ampicillin. Multi-drug resistance rate was low (21%) and mostly from broiler flocks (93%). All isolates were genotyped into 32 different PFGE genotypes (PT) and 34 different MLVA genotypes (MT). The dominant genotypes were PT6 (12.5%) and MT22 (50%). In specific sample groups, there was a correlation between genotypes, breeding type, geographic location, and isolation years of the isolates. There was no significant difference in the discrimination power of PFGE and MLVA. However, MLVA was more suitable for large sample groups and routine genotyping because it was easier, quicker, and less labor-intensive to use.

Genomic characterization of two carbapenem-resistant Serratia marcescens isolates causing bacteremia: Emergence of KPC-2-encoding IncR plasmids.

The occurrence and transmission of carbapenemase-producing-Enterobacterales (CPE) on a global scale has become a major issue. Clinical reports are rarely providing information on the genomic and plasmid features of carbapenem-resistant Serratia marcescens. Our objective was to investigate the resistance and transmission dynamics of two carbapenem-resistant S. marcescens that are resistant to carbapenem and have caused bacteremia in China. Blood specimens were taken from two individuals with bacteremia. Multiplex PCR was employed to identify genes that code for carbapenemase. Antimicrobial susceptibility tests and plasmid analysis were conducted on S. marcescens isolates SM768 and SM4145. The genome of SM768 and SM4145 were completely sequenced using NovaSeq 6000-PE150 and PacBio RS II platforms. Antimicrobial resistance genes (ARGs) were predicted using the ResFinder tool. S1 nuclease pulsed-field gel electrophoresis (S1-PFGE) and southern blotting were employed to analyze plasmids. Two S. marcescens that produced KPC-2 were identified from bloodstream infections. The antimicrobial susceptibility testing demonstrated that both of the isolates had a resistance to various antibiotics. The whole-genome sequence (WGS) and plasmid analysis revealed the presence of bla KPC-2-bearing IncR plasmids and multiple plasmid-borne antimicrobial resistance genes in the isolates. Our comparative plasmid analysis suggested that the two IncR plasmids identified in this study could be derived from a common ancestor. Our findings revealed the emergence of bla KPC-2-bearing IncR plasmid in China, which could be a hindrance to the transmission of KPC-2-producing S. marcescens in clinical settings.

Phenotypic and genetic alterations of Burkholderia pseudomallei in patients during relapse and persistent infections.

The bacterium Burkholderia pseudomallei is the causative agent of melioidosis, a severe tropical disease associated with high mortality and relapse and persistent infections. Treatment of melioidosis requires prolonged antibiotic therapy; however, little is known about relapse and persistent infections, particularly the phenotypic and genetic alterations of B. pseudomallei in patients. In this study, we performed pulsed-field gel electrophoresis (PFGE) to compare the bacterial genotype between the initial isolate and the subsequent isolate from each of 23 suspected recurrent and persistent melioidosis patients in Northeast Thailand. We used whole-genome sequencing (WGS) to investigate multilocus sequence types and genetic alterations of within-host strain pairs. We also investigated the bacterial phenotypes associated with relapse and persistent infections, including multinucleated giant cell (MNGC) formation efficiency and intracellular multiplication. We first identified 13 (1.2%) relapse, 7 (0.7%) persistent, and 3 (0.3%) reinfection patients from 1,046 survivors. Each of the 20 within-host strain pairs from patients with relapse and persistent infections shared the same genotype, suggesting that the subsequent isolates arise from the infecting isolate. Logistic regression analysis of clinical data revealed regimen and duration of oral antibiotic therapies as risk factors associated with relapse and persistent infections. WGS analysis demonstrated 17 within-host genetic alteration events in 6 of 20 paired isolates, including a relatively large deletion and 16 single-nucleotide polymorphism (stocktickerSNP) mutations distributed across 12 genes. In 1 of 20 paired isolates, we observed significantly increased cell-to-cell fusion and intracellular replication in the second isolate compared with the initial isolate from a patient with persistent infection. WGS analysis suggested that a non-synonymous mutation in the tssB-5 gene, which encoded an essential component of the type VI secretion system, may be associated with the increased intracellular replication and MNGC formation efficiency of the second isolate of the patient. This information provides insights into genetic and phenotypic alterations in B. pseudomallei in human melioidosis, which may represent a bacterial strategy for persistent and relapse infections.

Increased copy number of 23S ribosomal RNA gene with point mutation in MRSA associated with linezolid resistance in a patient treated with long-term linezolid

BackgroundMethicillin-resistant Staphylococcus aureus (MRSA) infection is one of the most difficult infections we have to treat. Linezolid is one of the effective treatment options for refractory MRSA infections. There are cases where we are forced to use long-term linezolid treatment for refractory MRSA infections. ObjectiveTo discuss the evolution of Linezolid resistance factors in clinical isolates of MRSA. MethodsWe investigated 16 MRSA isolated from a patient treated with linezolid for a long period of 75 days. We performed antibiotic susceptibility test, 23S rRNA genes sequencing analysis, Pulsed-field gel electrophoresis. ResultsMRSA isolates were susceptible to linezolid before the start of treatment, but became less susceptible by prolonged treatment. The 23S rRNA sequencing analysis of linezolid-resistant strains that appeared 17 days after the start of treatment with linezolid revealed that all resistant MRSA had the G2576T substitution (Escherichia coli 23S rRNA gene number). The number of copies of this mutation increased with the use of linezolid. ConclusionLong-term use of linezolid in a patient or reuse of linezolid in a patient who has been previously treated with linezolid can lead to the emerging of linezolid-resistant MRSA in the host.