Repetitive Sequence Polymerase Chain Reaction Research Articles

DNA methylation status of interspersed repetitive sequences in patients with migraine.

To analyse the methylation status of the Long Interspersed Nuclear Element-1 (LINE-1) and Short Interspersed Nuclear Element Alu (Alu) of peripheral blood mononuclear cells (PBMCs) from patients with migraine compared with healthy control subjects. This case-control study recruited patients with migraine without aura and age-matched healthy control subjects. PBMCs were purified from peripheral blood samples. Methylation levels and patterns of LINE-1 and Alu sequences were evaluated using combined bisulfite restriction analysis-interspersed repetitive sequences polymerase chain reaction. A total of 84 patients with migraine and 82 age-matched healthy controls were enrolled in the study. High levels of unmethylated cytosines in both the LINE-1 and Alu repetitive elements were observed in the migraine group compared with the control subjects. In addition, a significant difference was detected in the methylation level of LINE-1 between TT and CC genotype groups of the methylenetetrahydrofolate reductase gene. These results suggest that analysis of epigenetic biomarkers in PBMCs may help to identify patients at a higher risk of migraine development.

Short Communication: Genetic diversity of walnut blight-associated bacteria from China using rep-PCR

Abstract. Zheng C, Zhu J, Yang X, Fu B. 2022. Short Communication: Genetic diversity of walnut blight-associated bacteria from China using rep-PCR. Biodiversitas 23: 5681-5686. Walnut blight is caused by the bacterium Xanthomonas arboricola pv. juglandis (Xaj), is one of the most serious diseases in walnut production around the world. Understanding the genetic diversity of walnut blight pathogen is of great significance to developing a scientific and reasonable prevention and control strategy. The rep-PCR (Repetitive Sequence Polymerase Chain Reaction) technique is a powerful tool for analyzing plant pathogens. In this paper, using three pairs of rep-PCR universal primers (ERIC, BOX, and REP), a genetic diversity analysis was carried out on walnut blight-associated bacteria mainly isolated from different counties of Hubei Province, China. A study of 60 walnut blight-associated bacteria isolated in 2016 found that they had a high genetic similarity among them. There is no clear correlation between the genetic diversity of these isolates and their geographical or tissue origins. Interestingly, these clusters can also distinguish copper-resistant isolates from sensitive ones. Therefore, rep-PCR is a powerful tool to study the population of walnut pathogenic bacteria.

Incidence and Molecular Characterization of Carbapenemase Genes in Association with Multidrug-Resistant Clinical Isolates of Pseudomonas aeruginosa from Tertiary Healthcare Facilities in Southwest Nigeria.

Pseudomonas aeruginosa, resistant to multiple antibacterial agents including carbapenems, is of great global public health concern. There is limited data available regarding incidence of Metallo-Beta Lactamase producing P. aeruginosa, their molecular basis of resistance in particular carbapenem resistance and any genetic relatedness among circulating clinical isolates in Southwest Nigeria. Four hundred and thirty P. aeruginosa isolates were collected from seven tertiary care hospitals (predominantly from wound, ear, and urinary tract infections) and verified by PCR targeting oprI and oprL. Antibiotic susceptibility using 16 selected antibiotics and MBL screening was performed. The integrons (class 1, 2 and 3) and carbapenemase genes- blaGES, blaNMC-A, blaBIC-1, blaSME, blaIMP, blaVIM, blaSPM, blaNDM, blaAIM, blaDIM, blaSIM, blaGIM, blaOXA-48, blaOXA-58 were detected by PCR and were sequenced. Quantitative real-time polymerase chain reaction was used to quantify expression levels of eight efflux pump genes, ampC cephalosporinase and outer membrane porin, oprD. The isolates were genotyped using Enterobacterial Repetitive Intergenic Consensus sequence Polymerase Chain Reaction (ERIC-PCR). Four hundred and thirty P. aeruginosa isolates were subjected to antibiotic susceptibility testing, revealing that 109 (25.4%) isolates were multidrug-resistant, 47 (10.9%) were extensively drug-resistant and 25 (5.8%) were pandrug-resistant. MBL was seen in 17.0% (73/430) isolates. MBL-encoding genes; blaVIM-5 and blaNDM-1 were detected in 86.3% (63/73) isolates, with blaVIM-5 and blaNDM-1 in 35.6% (26/73) and 38.4% (28/73), respectively, whereas co-occurrence of blaVIM-5 and blaNDM-1 was found in 12.3% (9/73). Forty-one (56.2%) carbapenem-resistant P. aeruginosa strains carried class 1 integrons, while co-occurrence of class 1 and 2 integrons was seen in 12.3%. qPCR results indicated that MexXY-OprM was highly expressed pump in 58.9%, ampC upregulated in 26.0%, while oprD porin was downregulated in 65.8% isolates. ERIC-PCR results suggest that carbapenem-resistant strains exhibit genetic heterogeneity. The high incidence of MBL-encoding genes and integrons in diversified clinical P. aeruginosa from southwestern Nigeria is of great concern. The co-occurrence of blaVIM-5 and blaNDM-1 as well as resistance in general manifesting a gradient based on genotypic variation suggests that there is a strong need for efficient surveillance programs and antibiotic stewardship.

Environmental Methicillin-resistant Staphylococcus aureus Contamination, Persistent Colonization, and Subsequent Skin and Soft Tissue Infection

The longitudinal association among persistent Staphylococcus aureus colonization, household environmental contamination, and recurrent skin and soft tissue infection (SSTI) is largely unexplored to date. To identify factors associated with persistent S aureus colonization and recurrent SSTI in households with children with community-associated methicillin-resistant S aureus (MRSA) SSTI. This 12-month prospective cohort study included 150 children with community-associated MRSA SSTI, 542 household contacts, and 154 pets enrolled from January 3, 2012, through October 20, 2015. A total of 5 quarterly home visits were made to 150 households in the St Louis, Missouri, region. Statistical analysis was performed from September 18, 2018, to January 7, 2020. Covariates used in S aureus strain persistence and interval SSTI models included S aureus colonization and contamination measures, personal hygiene and sharing habits, health history, activities external to the home, and household characteristics (eg, cleanliness, crowding, home ownership, and pets). Serial samples to detect S aureus were collected from household members at 3 anatomic sites, from pets at 2 anatomic sites, and from environmental surfaces at 21 sites. Molecular epidemiologic findings of S aureus isolates were assessed via repetitive-sequence polymerase chain reaction. Individual persistent colonization was defined as colonization by an identical strain for 2 consecutive samplings. Longitudinal, multivariable generalized mixed-effects logistic regression models were used to assess factors associated with persistent S aureus personal colonization, environmental contamination, and interval SSTI. Among 692 household members in 150 households, 326 (47%) were male and 366 (53%) were female, with a median age of 14.82 years (range, 0.05-82.25 years). Of 540 participants completing all 5 samplings, 213 (39%) were persistently colonized with S aureus, most often in the nares and with the strain infecting the index patient at enrollment. Nine pets (8%) were persistently colonized with S aureus. Participants reporting interval intranasal mupirocin application were less likely to experience persistent colonization (odds ratio [OR], 0.44; 95% credible interval [CrI], 0.30-0.66), whereas increasing strain-specific environmental contamination pressure was associated with increased individual persistent colonization (OR, 1.17; 95% CrI, 1.06-1.30). Strains with higher colonization pressure (OR, 1.47; 95% CrI, 1.25-1.71) and MRSA strains (OR, 1.57; 95% CrI, 1.16-2.19) were more likely to persist. Seventy-six index patients (53%) and 101 household contacts (19%) reported interval SSTIs. Individuals persistently colonized with MRSA (OR, 1.56; 95% CrI, 1.17-2.11), those with a history of SSTI (OR, 2.55; 95% CrI, 1.88-3.47), and index patients (OR, 1.54; 95% CrI, 1.07-2.23) were more likely to report an interval SSTI. The study findings suggest that recurrent SSTI is associated with persistent MRSA colonization of household members and contamination of environmental surfaces. Future studies may elucidate the effectiveness of specific combinations of personal decolonization and environmental decontamination efforts in eradicating persistent strains and mitigating recurrent SSTIs.

Antibiotic Resistance And Genotyping Of Gram-Positive Bacteria Causing Hospital-Acquired Infection In Patients Referring To Children's Medical Center.

IntroductionA serious problem affecting human society is the development of bacterial resistance. The purpose of the current study was to evaluate the antibiotic resistance of Gram-positive bacteria (GPB) and genotyping of common GPB causing hospital-acquired infections (HAIs) in patients who were referred to Children’s Medical Center during a 6-month period by random amplified polymorphic DNA (RAPD) and enterobacterial repetitive intergenic consensus sequence polymerase chain reaction (ERIC-PCR).MethodsDuring the 6-month period, antimicrobial resistance profiles of GPB isolates recovered from patients in Children’s Medical Center were determined using the Kirby–Bauer disk diffusion and MIC. Typing of common GPB was performed and the results were analyzed by gel compare software.ResultsIn this cross-sectional study, 6524 cultures were performed and 138 Ggram-positive bacteria were isolated (2%). Staphylococcus aureus strains showed the highest antibiotic penicillin resistance (96.3%). Twenty-six per cent of the strains were methicillin-resistant S. aureus (MRSA) and no resistance was found against vancomycin. All isolates of Enterococcus faecium were resistant to ciprofloxacin (100%). The resistance to vancomycin was very high (67%) and no resistance was observed to linezolid. The results of genotyping analysis of S. epidermidis strains showed the presence of two clones with a genetic relationship of over 80%. All of the S. aureus strains were in one cluster and half of the E. faecium strains were in a cluster with a genetic predilection of over 80%.ConclusionThis study indicated frequent occurrence of antimicrobial resistance, especially in Enterococcus spp. isolates. Rapid spreads of MRSA and VREF from a clonal origin require implementing careful isolation and infection control measures.

Enterotoxin gene profile and molecular epidemiology of Aeromonas species from fish and diverse water sources.

This investigation was undertaken to study the prevalence, enterotoxin gene profile and molecular epidemiology of Aeromonads from various sources of water (182) and fish (173). A total of 116 Aeromonas sp. were isolated, of which 48 (26·37%) were from water and 68 (34·62%) were from fish samples collected from retail markets and fish farms. The Aeromonads were recovered from all types of water sources viz. drinking water (13%), surface waters (26%) and fish ponds (69%). The most prevalent species recovered from drinking water was A. hydrophila, from fish ponds it was A. caviae, from surface water sources A. hydrophila and A. caviae were recovered more frequently, and A. hydrophila and A. veronii bv. sobria were isolated predominantly from gills of fish samples. On multiplex PCR analysis for the detection of enterotoxin genes (act, alt, ast), the above mentioned Aeromonas species frequently contained enterotoxin genes, irrespective of their sources. From isolates across all the sources, act (63%) and alt (57%) genes were encountered more frequently than ast (6%). The enterobacterial repetitive intergenic consensus sequences polymerase chain reaction was used for typing of isolates and most of the isolates from water and fish were related, owing to similar ecosystem. A wide distribution of enterotoxin genes in Aeromonads from water and fish is a potential public health threat and molecular genotyping can be helpful to study epidemiology of the pathogen. A high proportion of isolates recovered from diverse water sources, particularly potable drinking water and fish samples carried one or more enterotoxin genes thereby indicating a potential pathogenic nature of isolates from these sources. The genetic relatedness was detected amongst many isolates recovered from water sources and fish samples indicating circulation of familiar virulent clones in the aquatic environments.

Prevalence and genetic diversity of Vibrio parahaemolyticus strains from salmon in Chinese markets.

Consumption of contaminated salmon in China has led to pathogenic Vibrio parahaemolyticus infections in humans. In this study, 420 salmon samples were collected from supermarkets and restaurants in China that showed a contamination rate of 9.05 and 15.24%, respectively. Eighteen antibiotics were used to test the antibiotic susceptibility of all 51 isolates. The most common resistance was observed to β-lactam antibiotics and aminoglycosides, including ampicillin (92.16%), streptomycin (88.24%), kanamycin (45.10%) and cephazolin (45.10%). Meanwhile, many strains were resistant to more than two antibiotics (48/51, 94.12%). Only two and five isolates were positive for tdh and trh, respectively. Serotyping results demonstrated that O2 was most prevalence serotype (15/51, 29.41%). Molecular typing (enterobacterial repetitive intergenic consensus sequence polymerase chain reaction and multilocus sequence typing) allowed classification of all the isolates into 5 clusters and 44 sequence types, highlighting genetic variation and relatedness. In general, the high antibiotic resistance is alarming and raises public health concerns. Frequent monitoring of salmon for V. parahaemolyticus contamination, genetic diversity and antibiotic susceptibility is essential to improve seafood safety.

Emergency Department Environmental Contamination With Methicillin-Resistant Staphylococcus aureus After Care of Colonized Patients

Methicillin-resistant Staphylococcus aureus (MRSA) transmission dynamics in the emergency department (ED) are not well defined; environmental surfaces may serve as reservoirs for transmission. This study investigates the effectof patients with a history of MRSA colonization or infection on subsequent MRSA contamination of the ED environment. Adult ED patients with evidence of an MRSA-positive surveillance result or clinical microbiologic culture in the year preceding their current ED visit were enrolled. Cultures from 5 anatomic sites were obtained to detect active MRSA colonization. After patients' discharge and before environmental disinfection, up to 16 prespecified surfaces in their ED rooms were cultured. Strain typing was performed by repetitive-sequence polymerase chain reaction on all recovered MRSA isolates to determine concordance with the corresponding patient strain. Of 42 patients enrolled, 25 (60%) remained colonized with MRSA. Nineteen of the 25 ED rooms (76%) occupied by MRSA-colonized patients contained greater than or equal to 1 MRSA-contaminated environmental surface on patient discharge. Surfaces were more likely to be contaminated when rooms were occupied by patients colonized with MRSA at 1 body site (odds ratio 11.7; 95% confidence interval 1.5 to 91.5) and greater than or equal to 2 body sites (odds ratio 16.3; 95% confidence interval 3.1 to 86.8) compared with noncolonized patients. In 16 of the 19 ED rooms (84%) where MRSA was recovered, all environmental strains were concordant with the corresponding patient strain. Contamination of the ED environment with MRSA from actively colonized patients is common. Improved environmental surface disinfection may help reduce transmission of MRSA to ED health care professionals and patients during emergency care.

Screening of tropical estuarine water in south-west coast of India reveals emergence of ARGs-harboring hypervirulent Escherichia coli of global significance.

The goal of this study was to investigate the involvement of a tropical Indian estuary in the emergence of antibiotic resistance genes (ARGs)-harboring hypervirulent E. coli of global significance. A total of 300 E. coli isolates was tested for antibiotic susceptibility to β-lactams, aminoglycosides, chloramphenicol, quinolones, sulphonamides, tetracyclines, and trimethoprim. The E. coli isolates were screened for the presence of antibiotic resistance genes (blaTEM, blaCTX-M, tetA, tetB, sul1, sul2, strA, aphA2, catI, dhfr1, and dhfr7), integrase (int1, int2, and int3), Shiga toxin genes (stx1 and stx2) and extraintestinal virulence genes (papAH, papC, sfa/focDE, kpsMT II, and iutA). The highest prevalence of antibiotic resistance was observed for ampicillin, followed by tetracycline, and nalidixic acid. Among E. coli isolates, 64% were resistant to at least one of the 15 antibiotics tested, and approximately 40% were multiple antibiotic-resistant (MAR). More than 40% (n = 122) of E. coli isolates had ARGs. Integrase 1 (int1) was found in 7.6% of E. coli isolates. Among E. coli isolates, 16.3% (n = 49) were extraintestinal pathogenic E. coli (ExPEC), and approximately 34.6% (n = 17) of ExPEC had ARGs. A hypervirulent ARGs-harboring STEC was isolated. The prevalence of Shiga toxin-producing E. coli (STEC) was low (n = 1). The prevalence of ARGs-harboring pathogenic E. coli isolates was higher in stations close to the City (urban area), than that of other stations. ERIC-PCR (enterobacterial repetitive intergenic consensus sequence polymerase chain reaction) analysis revealed a high degree of genetic diversity among the ARGs-harboring E. coli isolates. The results demonstrate a high prevalence of ARGs-harboring E. coli in estuarine water and confirm the need for a better wastewater treatment facility and proper control measures to reduce the discharge of sewage and wastewater into the aquatic environments.

Distribution and Genotyping of Aquatic Acinetobacter baumannii Strains Isolated from the Puzi River and Its Tributaries Near Areas of Livestock Farming

Acinetobacter baumannii is an important health care-associated bacterium and a common multidrug-resistant pathogen. The use of antibiotics in the husbandry industry has raised concerns about drug-resistant A. baumannii strains, which may affect humans. This study aimed to investigate the seasonal distribution of A. baumannii in aquatic environments near areas of livestock farming. The geographic distribution, antibiotic resistance characteristic, and DNA fingerprinting genotype of A. baumannii were also studied. The results showed that environmental A. baumannii was prevalent during the summer and autumn. The hotspots for A. baumannii were found at the sampling sites of livestock wastewater channels (21.4%; 3/14) and the tributaries adjacent to livestock farms (15.4%; 2/13). The prevalence of A. baumannii at these locations was significantly higher than those adjacent to the Puzi River. Multidrug-resistant strain of A. baumannii was not found in this study, with only one strain (5%; 1/20) being resistant to tetracycline. Of the isolates that were obtained, 10% (2/20) and 20% (4/20) were found to be intermediately resistant to tetracycline and sulphamethoxazole/trimethoprim, respectively. The genotyping patterns and clustering analysis indicated that enterobacterial repetitive intergenic consensus sequence polymerase chain reaction (ERIC-PCR) differentiated A. baumannii strains effectively. There were two major clusters that could then be subtyped into 20 A. baumannii strains with 15 profiles. The A. baumannii strains that were isolated from upstream of the Puzi River and livestock wastewater channels were composed of Cluster I. Cluster II only contained isolates from downstream of the Puzi River area. Furthermore, isolates from adjacent sites were shown to have identical profiles (100%). These results suggest that A. baumannii may have spread through free-flowing water in this study. Therefore, we propose that livestock wastewater is one of the sources that contribute to A. baumannii pollution in water bodies. In summary, continuous monitoring of antibiotic pollution in livestock wastewater is required.

Phenotypic and genotypic characterization of Clavibacter michiganensis subsp. michiganensis causing tomato bacterial canker and wilt disease in Turkey

Tomato bacterial canker and wilt disease caused by Clavibacter michiganensis subsp. michiganensis (Cmm) is among one of the major bacterial diseases associated with tomato (Solanum lycopersicum L.) in the western Mediterranean region of Turkey. A total of 118 Cmm isolates were obtained from the petiole and the main vein of leaves of different cultivars of diseased tomato plants, and these isolates were cultured in semiselective medium (mSCM). The identity of Cmm isolates was confirmed through gas chromatography-fatty acid methyl-esters (GC-FAME) analysis and polymerase chain reaction (PCR) using the primers, CMM5 and CMM6. The fatty acid analysis of all the Turkish isolates yielded major components that included anteisoheptadeconic acid (a15:0), palmitic acid (i16:0) and anteisoheptadeconic acid (a17:0); the analysis detected and categorized all the isolates into 10 different FAME groups. Among repetitive element sequence PCR (rep-PCR) analysis, Box primer yielded the most reproducible genomic profiles with band sizes that ranged from ~200 bp to 2 kb. The isolates were also separated into 12 groups by pulsed-field gel electrophoresis (PFGE) after digesting the total genomic DNA with SpeI, a rare cutting enzyme. The genome sizes of the different strains of Cmm were also determined after running unrestricted total genomic DNA, which yielded average values between 3.0 and 3.5 MB. All the Cmm isolates had pCM1 and pCM2 plasmids. This is the first report on the detailed characterization of the Cmm population in Turkey.

High Prevalence of Plasmid-Mediated Quinolone Resistance Determinants in Escherichia coli and Klebsiella pneumoniae Isolates from Pediatric Patients in China.

In this study, the prevalence of the plasmid-mediated quinolone resistance (PMQR) genes among pediatric clinical isolates of Escherichia coli and Klebsiella pneumoniae was investigated. A total of 243 nonduplicate clinical isolates of E. coli and K. pneumoniae collected between January 2010 and December 2012 from Beijing, China, were studied. In total, 55 isolates (22.63%) were positive for PMQR genes, the most frequently detected gene was qnrS (13.2%), followed by aac(6')-Ib-cr (6.2%) and qnrB (3.7%). The qnrA and qepA genes were not detected. Furthermore, 92.73% (51/55) produced extended-spectrum β-lactamases (ESBLs) and 21.82% (12/55) were resistant to quinolones. DNA sequencing results showed that 14.55% (8/55) of isolates possessed gyrA mutations, while 1.82% (1/55) had parC mutations in the quinolone resistance-determining region (QRDR). Nalidixic acid or ciprofloxacin resistance of the transconjugants increased from 2- to 32-fold. Enterobacterial repetitive intergenic consensus sequence polymerase chain reaction typing indicated that most isolates were not clonally related. Our findings showed that the PMQR detection rate among pediatric clinical isolates of E. coli and K. pneumoniae was high in China. PMQR-positive strains were more common among ESBL-producing and ciprofloxacin-susceptible isolates. Conjugation experiments showed that these isolates could be transferred horizontally. The present study highlights the high prevalence of PMQR in Chinese pediatrics who are not treated with quinolones.

Operational taxonomic units (OTUs) of endophytic bacteria isolated from banana cultivars in the Amazon.

Endophytic microorganisms colonize plants, inhibit the growth of pathogens (by competing for nutrients and/or space), or produce antagonistic substances. Fifty-five endophytic bacteria were isolated from the leaf tissue of the FHIA 18 banana cultivar. Genetic diversity analyses were performed using the enterobacterial repetitive intergenic consensus sequence polymerase chain reaction method and BOX molecular markers. These analyses resulted in 33 and 21 polymorphic bands, respectively. The similarity data, obtained using the Dice coefficient based on the polyphasic analysis method, ranged from 22 to 100%. This indicated a high genetic diversity among the analyzed isolates. Sixty percent similarity was utilized as the cut-off criterion for the formation of operational taxonomic units (OTUs); this resulted in the identification of 32 possible OTUs, indicating a high number of potential species.

Contamination of environmental surfaces with Staphylococcus aureus in households with children infected with methicillin-resistant S aureus.

Household environmental surfaces may serve as vectors for the acquisition and spread of methicillin-resistant Staphylococcus aureus (MRSA) among household members, although few studies have evaluated which objects are important reservoirs of MRSA. To determine the prevalence of environmental MRSA contamination in households of children with MRSA infection; define the molecular epidemiology of environmental, pet, and human MRSA strains within households; and identify factors associated with household MRSA contamination. Fifty children with active or recent culture-positive community-associated MRSA infection were enrolled from 2012 to 2013 at St Louis Children's Hospital and at community pediatric practices affiliated with the Washington University Pediatric and Adolescent Ambulatory Research Consortium in St Louis, Missouri. Samples of participants' nares, axillae, and inguinal folds were cultured to detect S aureus colonization. Samples of 21 household environmental surfaces, as well as samples obtained from pet dogs and cats, were cultured. Molecular typing of S aureus strains was performed by repetitive-sequence polymerase chain reaction to determine strain relatedness within households. Methicillin-resistant S aureus was recovered from samples of environmental surfaces in 23 of the 50 households (46%), most frequently from the participant's bed linens (18%), television remote control (16%), and bathroom hand towel (15%). It colonized 12% of dogs and 7% of cats. At least 1 surface was contaminated with a strain type matching the participant's isolate in 20 households (40%). Participants colonized with S aureus had a higher mean (SD) proportion of MRSA-contaminated surfaces (0.15 [0.17]) than noncolonized participants (0.03 [0.06]; mean difference, 0.12 [95% CI, 0.05-0.20]). A greater number of individuals per 1000 ft 2 (93 m2) were also associated with a higher proportion of MRSA-contaminated surfaces (β = 0.34, P = .03). The frequency of cleaning household surfaces was not associated with S aureus environmental contamination. Methicillin-resistant S aureus strains concordant with infecting and colonizing strains are present on commonly handled household surfaces, a factor that likely perpetuates MRSA transmission and recurrent disease. Future studies are needed to determine methods to eradicate environmental contamination and prevent MRSA transmission in households.

Clinical and Molecular Epidemiology of Pretransplant Vancomycin-Resistant Enterococci (VRE) Colonization Among Patients Who Undergo Liver Transplantation.

Purpose: To identify risk factors and outcomes associated with pre-liver transplant (LTx) VRE colonization and describe the molecular diversity among VRE strains colonizing patients who undergo LTx. Methods: An observational cohort study was conducted from 2010-2012. Peri-rectal VRE surveillance cultures were performed prior to LTx. Patients with pre-LTx VRE colonization were placed in contact isolation and received daptomycin in addition to routine perioperative antibiotic prophylaxis at LTx. Repetitive sequence polymerase chain reaction (rep-PCR) was used to determine clonality among colonizing strains. Results: Of 61 patients who underwent pre-LTx VRE surveillance and subsequent LTx 27 (44%) were colonized with VRE. Increased hospitalization days, intensive care days, central venous catheterization, higher MELD score, and prior antibiotic exposure were associated with pre-LTx VRE colonization. Three patients developed post-LTx VRE infections, all were VRE-colonized pre-LTx. Pre-LTx VRE colonization was associated with longer post-LTx hospitalization (p = 0.04). Among VRE isolates that underwent rep-PCR testing 15/22 (68%) had a similar molecular fingerprint pattern. Conclusions: Active VRE surveillance among LTx candidates identifies VRE-colonized patients who may benefit from targeted antimicrobial prophylaxis and enhanced infection prevention measures including contact isolation to control institutional VRE spread. A risk factor-based approach focusing on patients with high MELD scores, prolonged hospitalization and prior antibiotic exposure may increase the yield of VRE surveillance in this population. The high percentage of similar VRE isolates may suggest linked transmission and acquisition during pre-LTx hospitalizations.Table: No Caption available.

Molecular Epidemiology of Methicillin-resistant Staphylococcus aureus Isolated in Serial Cultures From the Respiratory Tract of Children with Cystic Fibrosis

Little is known about strain relatedness of methicillin-resistant Staphyloccocus aureus (MRSA) isolated at serial time points from the respiratory tract of patients with cystic fibrosis (CF). The objectives are to interrogate the genetic diversity of MRSA recovered in serial cultures from children with CF and to correlate strain relatedness with clinical characteristics. We performed a retrospective analysis of children with CF from whom MRSA was isolated from serial respiratory cultures from 2005 to 2011. Within individual patients, relatedness of isolated strains was determined by repetitive-sequence polymerase chain reaction, and the staphylococcal cassette chromosome mec type of each isolate was characterized. Medical records corresponding to the MRSA cultures were reviewed. We identified 54 CF patients with serial MRSA cultures (145 distinct cultures). Over time, 45 (83%) patients maintained the same strain type and 9 (17%) possessed at least 2 distinct strain types. A total of 91 pairs of isolates were analyzed for strain relatedness. Of these, 81 (89%) were identical and 10 (11%) were distinct strain types. About 117 (83%) isolates were staphylococcal cassette chromosome mec type II, 24 (17%) were staphylococcal cassette chromosome mec type IV and 4 were other types not resolvable with our assay. Clinical factors, including time interval and prescription of antibiotics effective against MRSA between positive cultures, did not correlate with acquisition of a distinct MRSA strain by individual patients. Our data suggest that sustained presence of MRSA in CF patients is most commonly attributable to identical strain types. Acquisition of distinct MRSA strains in the airway is infrequent.

Shiga-toxin genes and genetic diversity of Escherichia coli isolated from pasteurized cow milk in Brazil.

This study evaluated the genetic similarity and prevalence of the stx1, stx2, eae, and ehxA genes in Escherichia coli isolated from pasteurized cow milk. Eighty-seven E. coli isolates from pasteurized cow milk from 22 dairies located in northwestern Paraná state, Brazil, were analyzed. Genetic similarity was evaluated using enterobacterial repetitive intergenic consensus sequence polymerase chain reaction (ERIC-PCR) and repetitive extragenic palindromic sequence PCR (REP-PCR). E. coli isolates were also analyzed by PCR to investigate the presence of the stx1, stx2, eae, and ehxA genes. ERIC-PCR and REP-PCR clustered 87 bacterial isolates in 76 and 81 genomic profiles, respectively. Both techniques revealed high genetic diversity among the E. coli isolates, confirming the possibility of their use in epidemiological studies. The stx1, stx2, eae, and ehxA virulence genes were not detected in E. coli isolates, indicating a low prevalence of Shiga toxin-producing E. coli in milk produced in the region studied. Knowledge about the presence of diarrheagenic Escherichia coli in pasteurized milk is important developing and implementing control measures in milk and dairy production.

A single genotype of Acinetobacter baumannii expresses multiple antibiotic susceptibility phenotypes

It is common practice to define a bacterium with unequal antibiotic susceptibility phenotypes as a different genotype. Here, we describe the utility of this practice for Acinetobacter baumannii. We determined antibiotic susceptibility for 19 clinical isolates of A baumannii. Repetitive sequence polymerase chain reaction method was used to determine genotypes. Although all isolates were of the same genotype, 79% were predicted to be different based on susceptibility patterns. The antibiotic susceptibility phenotype does not accurately predict the genotype of A baumannii.

Molecular Typing of Cronobacter Strains from Food in China by Enterobacterial Repetitive Intergenic Consensus Sequence PCR (ERIC‐PCR) and Sequence Analysis of the gyrB Gene

AbstractThe occurrence of outbreaks of Cronobacter strains causing necrotizing meningitis in China highlights the need for strain characterization and subtyping of this pathogenic species. A total of 43 Cronobacter isolates and five Enterobacteriaceae strains were used in this study. The strains were characterized using two molecular typing methods, including enterobacterial repetitive intergenic consensus sequence polymerase chain reaction (ERIC‐PCR) and sequence analysis of the gyrB gene. Genetic profiles of cluster analysis from these two molecular typing tests clearly showed that there was no close relationship between Cronobacter spp. and Enterobacteriaceae strains. Discriminatory index of ERIC‐PCR typing for the 43 Cronobacter isolates and five Enterobacteriaceae strains was high (D = 0.984) by Simpson's index of diversity based on the similarity value of more than 80% using BioNumerics version 5.0. Sequence analysis of the gyrB gene was proven to be suitable for the phylogenetic analysis of the Cronobacter strains. These results demonstrate that ERIC‐PCR combined with sequence analysis of the gyrB gene may be a reliable, rapid typing strategy for Cronobacter strains.Practical ApplicationsMolecular typing of foodborne pathogenic bacteria has been shown as a useful tool for tracking the source of infection and detection of virulent strains, as well as for determining the geographical and host distribution of possible variants. ERIC‐PCR provides rapid clustered results when a large number of Cronobacter spp. isolates need to be subtyped rapidly. Furthermore, partial sequence analysis of the gyrB gene was utilized to differentiate among the closely related isolates. The results of the current study suggest that ERIC‐PCR in conjunction with the gyrB sequence analysis would provide a reliable and accurate typing strategy for Cronobacter spp. strains. This combination of techniques can be used as a rapid means of comparing Cronobacter spp. isolates for epidemiological investigations.

경기도내에서 분리한 캠필로박터 제주니균의 유전적특성 및 항생제내성 연구

Campylobacter jejuni는 사람에서 매우 중요한 식중독 원인균의 하나이며, 2010년 경기도내 병원을 내원한 설사 환자와 4번의 식중독 outbreak에서 42균주를 분리하였다. 본 연구에서는 분리된 C. jejuni 42균주의 유전적 특성과 혈청형, 항균제 내성율을 분석하였다. hipO 종특이 유전자(100%), cdtB 독소 유전자(100%)가 검출되었고, gyrA 돌연변이 유전자(95.2%)가 PCR 실험결과 검출되었다. gyrA 돌연변이 유전자는 ciprofloxacin 내성과 연관이 깊으며, 디스크 확산법에 의해 실험한 결과 gyrA 돌연변이 유전자가 검출된 40균주(95.2%)에서 실제 ciprofloxacin에 대해 내성을 보였다. 분리된 42균주 C. jejuni에 대한 gyrA 유전자의 염기서열을 분석한 결과 ciprofloxacin에 내성을 가진 40균주에서 아미노산 서열이 ACA (트레오닌)에서 ATA (이소루신)으로 돌연변이 되어있음을 확인하였다. 하지만 대체 치료제인 erythromycin과 azithromycin에 대해서는 97.6% (41균주) 감수성을 보였다. 또한 C. jejuni의 혈청형을 분석한 결과 4가지 타입으로 분류되었는데, HS2(B), HS3(C), HS4(D), HS19(O)로 확인되었다. 도내에서 분리한 C. jejuni 42균주의 유전형을 rep-PCR과 PFGE로 확인한 결과 PFGE에 의해서는 12 cluster, rep-PCR에 의해서는 11 cluster의 유전형으로 구분되었다. Campylobacter jejuni is one of important food-borne pathogens causing human gastroenteritis. We isolated 42 strains of C. jejuni from diarrhea patients and 4 food-poisoning outbreaks in 2010, Gyeonggi-do. In this study, 42 strains were tested for genetic characteristics, the serotype distribution and antimicrobial resistant rate. The presence of hipO (100%), cdtB (100%), and mutated gyrA (95.2%) genes was detected in C. jejuni by polymerase chain reaction (PCR). Detection of mutated gyrA gene correlated with ciprofloxacin resistance. Forty isolates had mutated gyrA gene and were actually resistant to ciprofloxacin. Furthermore, comparing the gyrA DNA sequence data, ciprofloxacin-resistant isolates had a mutation of the DNA sequence from ACA (threonine) to ATA (isoleucine). But 41 strains (97.6%) of patient isolates were susceptible to erythromycin and azithromycin. A total of 35.7% among 42 C. jejuni isolates were identified into 4 different serotypes. The serotype distribution of C. jejuni strains were shown to be HS2(B), HS3(C), HS4(D), HS19(O). To investigate the genotypes of C. jejuni isolated in Gyeonggi province, repetitive sequence polymerase chain reaction (rep-PCR) analysis and SmaI-digested pulsed-filed gel electrophoresis (PFGE) profile analysis were performed. From the PFGE analysis of 42 C. jejuni strains, 12 clusters of PFGE profile were obtained. On the other hand, 11 clusters of rep-PCR profile were obtained from 42 strains of C. jejuni.