Enterobacterial Repetitive Intergenic Consensus Sequence PCR Research Articles

Polymerase chain reaction-based typing methods and protein profiling analysis of Acinetobacter baumannii isolated from environmental and clinical sources from South India.

Acinetobacter baumannii is an opportunistic pathogen known for causing hospital-acquired infections. The natural habitat includes soil, water, sewage, and drains, but it is also detected in infected individuals' blood, pus, and respiratory pathways. Due to its resilient nature, it is known to be a causative agent for outbreaks. Therefore, it is crucial to understand the genetic similarity between clinical and environmental isolates. The study aimed to find the genetic relationships between clinical and environmental isolates using PCR-based typing methods such as enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR), random amplified polymorphic DNA (RAPD), and repetitive extragenic palindromic sequence-based PCR (Rep-PCR). Additionally, outer membrane protein (OMP) and whole cell protein (WCP) profiles were also used. The PCR-based methods, ERIC-PCR and Rep-PCR, showed decreased genetic similarity between clinical and environmental isolates (66% and 58%, respectively). However, RAPD showed relatively higher genetic similarity (91%). The OMP and WCP profiles showed varied banding patterns between the clinical and environmental isolates in the 29-43 kDa region. The PCR-based methods proved to be a reliable and reproducible technique. The OMP and WCP profiles, though not as discriminatory as the molecular typing methods, could help identify the most and least commonly occurring protein bands and thus help in typing clinical and environmental A. baumannii isolates.

Repetitive extragenic palindromic and enterobacterial repetitive intergenic consensus sequence‐based typing of Shiga toxin‐producing Escherichia coli from bovine samples

AbstractThe strain typing of Shiga toxin‐producing Escherichia coli (STEC) is challenging due to its high genetic variability. In the present study, two polymerase chain reaction (PCR)‐based typing methods—repetitive extragenic palindromic sequences PCR (REP‐PCR) and enterobacterial repetitive intergenic consensus sequences PCR (ERIC‐PCR)—are evaluated for their efficacy in differentiating the strains. The present study used 100 strains of STEC isolated from bovine meat, bovine feces, and swabs from the chopping boards. The REP‐PCR profiles showed 21 different banding patterns, whereas ERIC‐PCR profiles obtained 18 patterns among the tested strains. The dendrogram prepared by the neighbor‐joining method formed eight and seven clusters for REP and ERIC‐PCR, respectively. The discriminatory indices for the typing methods were 87.83 and 83.03% for REP and ERIC‐PCR, respectively. Thus, the clusters based on REP and ERIC‐PCR fingerprints were unambiguous and thus could be useful for phylogenetic clustering and epidemiological surveillance. The nonparametric multidimensional scaling plots showed more than 75 and 60% similarities within populations of different REP and ERIC clusters, respectively, regarding its source of isolation, serogroup, and distribution virulence markers. The REP and the ERIC sequences are good molecular markers that offer rapid, cost‐effective, and easy molecular typing for STEC strains.

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.

Diversity of Vibrio parahaemolyticus in marine fishes of Bangladesh.

To determine the occurrence, diversity, antibiotic resistance and biofilm formation of Vibrio parahaemolyticus isolated from marine fishes in Bangladesh. A total of 80 marine fishes were obtained from the local markets and examined for the presence of V. parahaemolyticus. All the isolated V. parahaemolyticus were characterized for the presence of virulence markers, thermostable direct hemolysin (TDH) or thermostable direct hemolysin related hemolysin (TRH). Isolates were serotyped and further characterized by enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) typing to analyse the genetic diversity. Moreover, biofilm formation and antibiotic resistance patterns were also determined. About 63·75% (51/80) of the tested marine fishes were contaminated with V. parahaemolyticus. From the contaminated fishes, 71 representatives V. parahaemolyticus were isolated and none of them harboured tdh and trh virulence genes. Nine different O-groups and seven different K-types were found by serological analysis and the dominant serotype was O5:KUT. In ERIC-PCR analysis, eight clusters (A-H) were found and the most common pattern was A (46·5%). All of the isolates were resistant to ampicillin and 78·9% of isolates were resistant to streptomycin. The highest biofilm formation was found at 37°C compared to 25°C and 4°C. Diverse V. parahaemolyticus are present in marine fishes in the local market of Bangladesh with antibiotic-resistant properties and biofilm formation capacity. The widespread prevalence of diverse V. parahaemolyticus in marine fishes is an issue of serious concern, and it entails careful monitoring to ascertain the safety of seafood consumers.

Fecal Carriage and Molecular Epidemiology of Carbapenem-Resistant Enterobacteriaceae from Inpatient Children in a Pediatric Hospital of Shanghai.

PurposeTo determine the epidemiology characteristics of intestinal colonization of carbapenem-resistant Enterobacteriaceae (CRE) among inpatients in a pediatric hospital in China.MethodsA retrospective study was conducted from April to December 2019. Medical records were reviewed to extract the clinical information. Antimicrobial susceptibility was performed by broth microdilution method. Drug resistance determinants and plasmid types were analyzed using polymerase chain reaction (PCR) assays. Multilocus sequence typing (MLST) and Enterobacterial repetitive intergenic consensus sequences PCR (ERIC-PCR) were employed to determine the genetic relationships between strains.ResultsA total of 90 CRE strains were isolated, with a fecal carriage rate of 8.6% (90/1052), and mainly distributed in E. aerogenes (n=30), K. pneumoniae (n=25) and E. coli (n=23). More than 50% of CRE colonizers had a history of invasive procedures and antibiotic exposures. As high as 91.1% (82/90) of CRE isolates carried carbapenemase genes, with blaNDM-5 (n=56) being the most common, and mainly found in E. aerogenes (51.8%, 29/56) and E. coli (32.1%, 18/56) isolates, which primarily belonged to ST4 (100%, 29/29) and ST692 (55.6%, 10/18), respectively. Followed by blaKPC-2 (n=12), and all found in K. pneumoniae ST11 isolates. Other carbapenemase genes including blaNDM-1, blaIMP-4 and blaIMP-26. Meanwhile, ESBL genes (blaCTX-M, blaTEM-1 and blaSHV) and AmpC genes (blaDHA-1 and blaEBC) were also detected. All CRE isolates showed high resistance to cephalosporins and carbapenemases (97.8%-100.0%) but remained susceptible to tigecycline (98.9%). IncX3 was a major plasmid type in NDM-containing strains (91.3%), and 91.7% of KPC-2-producing K. pneumoniae harboring IncFII and IncFIB plasmids. The ERIC-PCR revealed that several strains with identical STs were genetically similar.ConclusionThis study revealed a major intestinal colonization of ST4 NDM-5 E. aerogenes, ST11 KPC-2 K. pneumoniae and ST692 NDM-5 E. coli strains among inpatients in a pediatric hospital. Infection control measures should be implemented immediately to prevent the spread of these strains in clinical settings.

First Identification and Characterization of Lactococcus garvieae Isolated from Rainbow Trout (Oncorhynchus mykiss) Cultured in Mexico.

Simple SummaryLactococcus garvieae is an emerging pathogen that causes lactococcosis, which is a septicemia disease that, as per reports, primarily affects rainbow trout and tilapia in various parts of the world. In rainbow trout, the clinical disease is very similar to other diseases caused by Gram-positive bacteria, including weissellosis, which has been previously reported in trout in Mexico. Following septicemia outbreaks that tested negative for weissellosis, a study was conducted to determine the etiology. This study confirms, for the first time, the presence of L. garvieae in processes of a septicemia disease affecting five rainbow trout farms in Mexico. Fourteen isolates were obtained from different geographical regions, but presented homogenous biochemical and protein profiles, as well as weak genetic heterogeneity. This study evidences the wide distribution and incidence of lactococcosis cases at rainbow trout farms in Mexico, demonstrating the need to develop vaccination programs against this disease.Lactococcosis is a hyperacute hemorrhagic septicemia disease caused by Lactococcus garvieae, which is an emerging pathogen in global fish farming. Between 2016 and 2018, rainbow trout (Oncorhynchus mykiss) from five farms that presented outbreaks were sampled as part of a Mexican surveillance program for the detection of fish diseases. Fourteen L. garvieae isolates were recovered from sampled fish, as confirmed by biochemical tests, 16S rRNA gene sequencing, and clinical and histological insights. The biochemical and protein profiles of the isolates obtained were homogeneous. Repetitive extragenic palindromic—(REP)—and enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) analyses established weak genetic heterogeneity. Rainbow trout challenged with two of the isolates used at different bacterial concentrations (10−2 and 10−4 CFU/mL) showed melanosis, and hemorrhages were observed in the fins, liver, kidney, and spleen. Isolates were obtained from all of the organs sampled, including from surviving fish, as either pure or mixed cultures. The present study is the first to confirm the presence of L. garvieae as the agent of severe lactococcosis outbreaks in the two primary Mexican states for trout farming.

Prevalence and antimicrobial resistance of retail-meat-borne Salmonella in southern China during the years 2009–2016: The diversity of contamination and the resistance evolution of multidrug-resistant isolates

Salmonella, one of the most important foodborne pathogens, can be the cause of bacterial food-borne illness and is commonly associated with the consumption of retail meat. Multidrug-resistant Salmonella isolates with high adaptability, have been responsible for many foodborne disease outbreaks. Here we present an investigation on the contamination and the antimicrobial resistance of Salmonella in retail meat obtained from supermarkets and from open markets in Guangxi, China. From the years 2009 to 2016, a total of 604 Salmonella isolates were recovered from a total of 3340 meat samples including 797 beef, 911 pork, 942 chicken and 690 duck, representing 18.08% of the samples tested. Pork was the most contaminated meat. Salmonella was detected in 322 samples from supermarkets and the positive rate of 21.03% was higher than that of 15.70% in 284 samples from open markets (P<0.05). The prevalence of Salmonella in retail meat in the summer and fall months: June (2015, 40.63%), October (2012, 34.6%; 2016, 43.75%) was higher than in other seasons of the year. One hundred and twenty-seven serotypes were identified among the 604 Salmonella enterica isolates, and S. Derby (28.48%), S. Agona (9.77%), S. London (4.97%) and S. Enteritidis (4.47%) were the most common serotypes. Tests of susceptibility to 21 antimicrobial agents showed that 87.58% of the isolates were resistant to at least one antimicrobial, and 57.79% exhibited multidrug resistance (MDR), as they were resistant to at least three antimicrobials. The presence of most of the antimicrobial-resistant genes tested was consistent with the resistant phenotypes found. Among all the antimicrobial resistant genes (ARGs) examined in this study, blaTEM-1, aadA1, cmlA, tetA, sul1 and sul2 were the most prevalent resistant genes in the multidrug resistant isolates. Our findings show that there was a trend that the Salmonella contamination in retail meat had increased and isolates showed an MDR phenotype and that the MDR had become more and more serious. Twenty-one isolates of S. Agona were randomly analyzed by using the enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) and six different types were found, indicating the existence of cross-contamination in the food market. The results indicate that the hazard analysis of the critical control points (HACCP) system for the whole food chain of retail meat should be further analyzed and improved.

Prevalence, virulence, antimicrobial resistance, and molecular characterization of fluoroquinolone resistance of Vibrio parahaemolyticus from different types of food samples in China

Vibrio parahaemolyticus is the leading cause of foodborne bacterial poisoning in China. The aim of this research is to conduct a study on the prevalence, virulence, and antimicrobial resistance of V. parahaemolyticus from different types of food samples in 12 different cities of China. Since fluoroquinolones are the major choice of treatment for V. parahaemolyticus infections, the genetic basis for fluoroquinolone resistance in V. parahaemolyticus were also investigated. V. parahaemolyticus was detected in 163 of the 784 food samples collected from 12 different cities in China, resulting in a prevalence of 20.79%. The prevalence of V. parahaemolyticus in ready-to-eat (RTE) food (4.96%) was much lower than those of shrimp (32.62%) and fish (22.00%). Virulence gene screening showed that 44 (27.00%) V. parahaemolyticus strains carried at least one virulence gene. Four isolates from shrimp and three isolates from fish contained both the virulence genes tdh and trh. In addition, the trh was firstly detected in one isolate collected from RTE food. All isolates exhibited relatively high resistance rates to ampicillin (82.21%), gentamicin (19.63%), and tetracycline (14.11%), while <10% of strains were resistant to ciprofloxacin (4.91%), levofloxacin (4.91%), and tetracycline (4.29%). Eight fluoroquinolone-resistant V. parahaemolyticus were selected to determine the molecular basis for fluoroquinolone resistance. These eight isolates belonged to three different types according to enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR). A Ser83Ile substitution in GyrA was deteted in seven fluoroquinolone-resistant strains, except V209 which harbored a Ser83Phe substitution in GyrA. Moreover, A Ser85Leu substitution in ParC was found in five isolates (V52, V53, V61, V163, and V209). Plasmid-mediated quinolone resistance (PMQR) genes were detected in all eight fluoroquinolone-resistant V. parahaemolyticus strains. This is the first report of Ser83Phe substitution in GyrA, qnrD and qnrS1 in V. parahaemolyticus. The information generated in this study will provide valuable information for risk assessment of V. parahaemolyticus infections and future control of antibiotic-resistant V. parahaemolyticus species in China.

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.

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.

Antibiotic susceptibility and molecular characterization of Aeromonas hydrophila from grass carp

AbstractAeromonas hydrophila is the leading cause of fish borne outbreaks and also represents a major threat to human health worldwide. This bacterium harbors many virulence factors such as aerolysin, elastase, and lipase genes. The extensive use of antibiotics is a contributing factor to the increasing incidence of antimicrobial‐resistant A. hydrophila. In the current study, we aimed to determine the incidence and features of A. hydrophila in grass carp in south China. We found 63 (31.5%) A. hydrophila isolates through investigation of 200 samples from five provinces in south China. All isolates were analyzed for virulence genes by polymerase chain reaction (PCR), antibiotic susceptibility analysis was carried out using the disk diffusion method, and molecular typing was performed using enterobacterial repetitive intergenic consensus sequence PCR (ERIC‐PCR). The results showed that the positive for aerolysin, elastase, hemolysin, and lipase gene were 71.43, 46.03, 0.00, and 55.56%, respectively. Antimicrobial susceptibility results indicated that most strains were resistant to ampicillin (95.24%), tetracycline (88.89%), and streptomycin (79.37%). A lot of multiple antibiotic resistance were found. The isolates were grouped into five clusters by ERIC‐PCR and shown the genetic diversity. Thus, our findings demonstrated the presence of A. hydrophila from grass carp, provided insights into the dissemination of antibiotic‐resistant strains, and improved our knowledge of microbiological risk assessment in this bacteria.Practical applicationsInvestigating the prevalence of Aeromonas hydrophilia isolates from grass carp could improved for assuring this microbial safety on food. In this study, antibiotic susceptibility, virulence genes, and molecular subtyping were selected to comprehensive analyze A. hydrophilia isolates. As grass carp are common and popular fish choices in China, therefore the continuous monitoring of this pathogens are vital to ensure the safety.

Antibiotic Susceptibility and Molecular Characterization of Escherichia coli O157 Isolates from Urinary Tract Infections

Background:Escherichia coli O157 is an important food-borne pathogen that can cause diarrhoea, hemorrhagic colitis, and hemolytic uraemic syndrome. Enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) and multilocus sequence typing (MLST) are good methods for molecular typing and the extensive use of antibiotics is a contributing factor to the increasing incidence of antimicrobial-resistant for these strains. Objectives: The aim of this study was to determine the prevalence, antimicrobial resistance, and genetic diversity of E. coli O157 based on the prevalence of urinary tract infection (UTI) in Hubei, China. Results: We obtained 23 (8.07%) E. coli O157 isolates from 285 UTI patients in Hubei, China. All isolates were subjected to antibiotic susceptibility analysis, and molecular typing was performed using ERIC-PCR and MLST. Antimicrobial susceptibility results indicated that most strains were resistant to penicillin (95.65%), chloramphenicol (73.91%), and ampicillin (69.57%). All isolates were discovered to be multiresistant (resistance to more than 3 antibiotics). Genetic variability analysis showed that all of the isolates were grouped into 4 clusters both by ERIC-PCR and MLST. Conclusion: Our findings demonstrated the presence of E. coli O157 in UTIs, provided insights into the dissemination of antibiotic-resistant strains, and improved our knowledge of E. coli O157 risk assessment in UTIs.

Discriminatory Power of Three Typing Techniques in Determining Relatedness of Nosocomial Klebsiella pneumoniae Isolates from a Tertiary Hospital in India

Purpose: The purpose of this study was to evaluate the discriminatory power of two DNA-based technique and a protein-based technique for the typing of nosocomial isolates of Klebsiella pneumoniae. A second objective was to determine the antimicrobial susceptibility pattern and characterise the presence of genes encoding extended-spectrum beta-lactamases (ESBLs) and carbapenemases. Materials and Methods: Forty-six K. pneumoniae isolates from patients with bloodstream infections at a tertiary care hospital in India between December 2014 and December 2015 were studied. All isolates were typed using enterobacterial repetitive intergenic consensus sequence-polymerase chain reaction (ERIC-PCR), randomly amplified polymorphic DNA (RAPD) analysis and matrix-assisted laser desorption ionisation time-of-flight (MALDI-TOF) mass spectrometry. Antimicrobial susceptibility profiles and ESBLs were detected using the BD Phoenix system. The types of ESBL and carbapenemase genes present were detected using PCR. Results: Isolates were subtyped into 31, 30 and 33 distinct genotypes by ERIC-PCR, RAPD and MALDI-TOF, respectively. Several isolates displaying identical DNA fingerprints were binned into different branches based on their proteomic fingerprint. Antimicrobial susceptibility tests revealed that 33/46 strains were multidrug resistant (MDR); a majority of the strains (83%) were sensitive to colistin. PCR-based analysis demonstrated 19 strains to harbour two or more ESBL and carbapenemase genes. Conclusion: ERIC-PCR was the most reproducible method for typing K. pneumoniae isolates and could not be substituted by MALDI-TOF for clonality analysis. A high degree of genetic diversity and the presence of MDR genes highlight the challenges in treating K. pneumoniae-associated infections.

Comparative isolation and genetic diversity of Arcobacter sp. from fish and the coastal environment.

Arcobacter sp. are emerging food- and water-borne human pathogens. In this study, comparison of two selective media suggested Arcobacter blood agar to be more efficient in yielding Arcobacter sp. from seafood. Furthermore, the isolation of Arcobacter sp. such as Arcobacter butzleri, A. skirrowii, A. mytili and A. defluvii from seafood suggests diverse sources of contamination of seafood by Arcobacter sp. Analysis of enterobacterial repetitive intergenic consensus sequence-PCR patterns of A. butzleri showed high genetic diversity and lack of clonality among the isolates. Arcobacter contamination of seafood is an emerging issue both from seafood safety and seafood trade point of view.

The prevalence and characterization of antibiotic-resistant and virulent Escherichia coli strains in the municipal wastewater system and their environmental fate

Antibiotics are widely used in human and veterinary medicine and in animal production, which increases their concentrations in aquatic ecosystems and contributes to selective pressure on environmental microorganisms. The objective of this study was to identify antibiotic resistance determinants in Escherichia coli strains isolated from untreated and treated wastewater (UWW and TWW) and from river water sampled downstream and upstream (URW and DRW) from the effluent discharge point. The analyzed antibiotic groups were beta-lactams, tetracyclines and fluoroquinolones which are widely used in human and veterinary medicine. The virulence of the isolated E. coli strains was also analyzed, and their clonal relatedness was determined by ERIC (enterobacterial repetitive intergenic consensus sequence) PCR. The highest counts of bacteria resistant to beta-lactams, tetracyclines and fluoroquinolones were noted in UWW at 6.4×104, 4.2×104 and 3.1×103CFU/mL, respectively. A total of 317 E. coli isolates resistant to at least one group of antibiotics were selected among bacterial isolates from river water and wastewater samples. Nearly 38% of those isolates were resistant to all of the tested antibiotics. The highest percent (43%) of multidrug-resistant isolates was noted in UWW samples. Isolates resistant to beta-lactams most frequently harbored blaTEM and blaOXA genes. The group of genes encoding resistance to tetracyclines was most frequently represented by tetA, tetB and tetK, whereas the qnrS gene was noted in isolates resistant to fluoroquinolones. Virulence genes bfpA (65%), ST (56%) and eae (39%) were most widely distributed in all isolates, regardless of their origin. The results of this experiment reveal the dangers associated with environmental contamination by drug-resistant and virulent E. coli strains distributed with treated wastewater. Multidrug resistance was determined more frequently in strains isolated from DRW than in isolates from URW samples. Our findings provide valuable inputs for evaluating public health hazards associated with bacterial contamination.

Diversity of Cronobacter spp. isolates from the vegetables in the middle-east coastline of China.

Cronobacter spp. has caused life-threatening neonatal infections mainly resulted from consumption of contaminated powdered infant formula. A total of 102 vegetable samples from retail markets were evaluated for the presence of Cronobacter spp. Thirty-five presumptive Cronobacter isolates were isolated and identified using API 20E and 16S rDNA sequencing analyses. All isolates and type strains were characterized using enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR), and genetic profiles of cluster analysis from this molecular typing test clearly showed that there were differences among isolates from different vegetables. A polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) based on the amplification of the gyrB gene (1258bp) was developed to differentiate among Cronobacter species. A new PCR-RFLP assay based on the amplification of the gyrB gene using Alu I and Hinf I endonuclease combination is established and it has been confirmed an accurate and rapid subtyping method to differentiate Cronobacter species. Sequence analysis of the gyrB gene was proven to be suitable for the phylogenetic analysis of the Cronobacter strains, which has much better resolution based on SNPs in the identification of Cronobacter species specificity than PCR-RFLP and ERIC-PCR. Our study further confirmed that vegetables are one of the most common habitats or sources of Cronobacter spp. contamination in the middle-east coastline of China.

Prevalence, Molecular Characterization, and Antibiotic Susceptibility of Vibrio parahaemolyticus from Ready-to-Eat Foods in China.

Vibrio parahaemolyticus is the leading cause of foodborne outbreaks, particularly outbreaks associated with consumption of fish and shellfish, and represents a major threat to human health worldwide. This bacterium harbors two main virulence factors: the thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH). Additionally, various serotypes have been identified. The extensive use of antibiotics is a contributing factor to the increasing incidence of antimicrobial-resistant V. parahaemolyticus. In the current study, we aimed to determine the incidence and features of V. parahaemolyticus in ready-to-eat (RTE) foods in China. We found 39 V. parahaemolyticus strains on Chinese RTE foods through investigation of 511 RTE foods samples from 24 cities in China. All isolates were analyzed for the presence of tdh and trh gene by PCR, serotyping was performed using multiplex PCR, antibiotic susceptibility analysis was carried out using the disk diffusion method, and molecular typing was performed using enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) typing and multilocus sequence typing (MLST). The results showed that none of the isolates were positive for tdh and trh. Most of the isolates (33.3%) were serotype O2. Antimicrobial susceptibility results indicated that most strains were resistant to streptomycin (89.7%), cefazolin (51.3%), and ampicillin (51.3%). The isolates were grouped into five clusters by ERIC-PCR and four clusters by MLST. We updated 10 novel loci and 33 sequence types (STs) in the MLST database. Thus, our findings demonstrated the presence of V. parahaemolyticus in Chinese RTE foods, provided insights into the dissemination of antibiotic-resistant strains, and improved our knowledge of methods of microbiological risk assessment in RTE foods.

Labrys soli sp. nov., isolated from the rhizosphere of ginseng.

In this study, we describe strain DCY64T that was isolated from the rhizosphere of three-year-old Korean ginseng root. Cells were Gram-reaction negative, oxidase- and catalase-positive, strictly aerobic, capsulated, non-motile, non-sporulating and spherical to short rod-shaped. Multiplicative budding cells were produced. Vesicles covered the surface of cells. Phylogenetic analysis placed strain DCY64T within the genus Labrys with the highest similarity to Labrys monachus VKM B-1479T (97.6 % 16S rRNA gene sequence similarity), followed by Labrys okinawensis MAFF 210191T (97.5 %), Labrys miyagiensis G24103T (97.4) and Labrys portucalensis F11T (97.0 %). The genomic DNA G+C content was 63 mol%. The presences of summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c), C19 : 1 cyclo ω8c and C16 : 0 as major fatty acids; phosphatidylmonomethylethanolamine, phosphatidylglycerol, phosphatidylcholine and diphosphatidylglycerol as major polar lipids; ubiquinone Q-10 as the predominant quinone and sym-homospermidine as the dominant polyamine were found in strain DCY64T. These chemotaxonomic results were in accordance with those of members of the genus Labrys. However, the absence of C16 : 0 2-OH, C16 : 0 3-OH and C18 : 1 2-OH from the fatty acids profile and differences in minor polar lipids and phenotypic characteristics distinguished strain DCY64T from the closest type strains. The discrimination was also supported by unique enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) fingerprints, as well as DNA-DNA hybridization values ( ≤ 48 %) between strain DCY64T and related type strains. Therefore, we propose that strain DCY64T represents a novel species of the genus Labrys. The name Labrys soli sp. nov. is proposed, with DCY64T ( = KCTC 32173T = JCM 19895T) as the type strain.

Prevalence and population analysis of Vibrio parahaemolyticus in aquatic products from South China markets.

Vibrio parahaemolyticus is a common foodborne pathogen in aquatic products. To investigate the prevalence of Vibrio parahaemolyticus in aquatic products in South China, 224 samples were collected from markets in four provinces (11 cities) from May 2013 to January 2014. One hundred and fifty isolates were isolated from 98 samples. All isolates were analyzed for the presence of thermostable direct haemolysin (TDH) and TDH-related haemolysin (TRH) by PCR, antibiotic susceptibility analysis by disk diffusion method, serotyping by multiplex PCR and molecular typing by enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) typing. Although all 150 isolates were negative for tdh, 61 strains were trh positive (40.67%). Antimicrobial susceptibility results indicated that most strains were resistant to streptomycin (88.67%), cefazolin (66.00%) and ampicillin (62.67%). All strains were susceptible to chloramphenicol. Forty percent of all isolates were O2 type. The 150 isolates were grouped into three clusters by ERIC-PCR typing. The results demonstrated the presence of V. parahaemolyticus in aquatic products from the retail market and this methodology can be used for microbiological risk assessment in China.