Repetitive Element Sequence-based PCR Research Articles

Fermentation Dynamics of Naturally Fermented Palm Beverages of West Bengal and Jharkhand in India

The term ‘toddy’ represents a group of different varieties of mild-alcoholic palm beverages of coastal and inland India, produced from the fresh saps of various palm trees through uncontrolled natural fermentation. In this study, we analysed the successional changes of microbial abundances and various physico-chemical parameters during natural fermentation (0 h to 48 h) of taal toddy, prepared from Palmyra palm, and khejur toddy, prepared from date palm of West Bengal and Jharkhand in India. Microorganisms from different successional levels were isolated and grouped using repetitive element sequence-based PCR (rep-PCR) technique and identified by the sequencing of 16S rRNA gene and D1-D2 region of 26S rRNA gene for bacteria and yeasts, respectively. Enterococcus faecalis, Lactiplantibacillus plantarum, Lacticaseibacillus paracasei and yeast Saccharomyces cerevisiae were identified during natural fermentation of toddy. During the natural fermentation, the average pH and total sugar content in the samples of both taal and khejur toddy decreased, whereas a gradual rise was observed in the contents of acidity, total alcohol, total ester and total protein. Bio-active potential (presence of phenolics and flavonoids) of toddy was also analysed (0 h to 48 h), where contents of total phenolics, flavonoids and resulting anti-oxidant activity were found higher in the end-product than the fresh palm sap, indicating toddy as a functional low-alcoholic drink. Lastly, it can be concluded that the inter-variable dynamics and microbial interrelation, which in turn depend on a number of local factors, regulate the overall fermentation dynamics and determine the product quality.

Identification and Characterization of Pseudomonas syringae pv. syringae, a Causative Bacterium of Apple Canker in Korea

In the present investigation, bacterial isolates from infected apple trees causing apple canker during winter were studied in the northern Gyeongbuk Province, Korea. The pathogen was identified as Pseudomonas syringae pv. syringae (Pss) through various physiological and biochemical characterization assays such as BIOLOG, gas chromatography of fatty acid methyl esters, and 16S rRNA. Bioassays for the production of phytotoxins were positive for syringopeptin and syringomycin against Bacillus megaterium and Geotrichum candidum, respectively. The polymerase chain reaction (PCR) method enabled the detection of toxin-producing genes, syrB1, and sypB in Pss. The differentiation of strains was performed using LOPAT and GATTa tests. Pss further exhibited ice nucleation activity (INA) at a temperature of -0.7°C, indicating an INA+ bacterium. The ice-nucleating temperature was -4.7°C for a non-treated control (sterilized distilled water), whereas it was -9.6°C for an INA- bacterium Escherichia coli TOP10. These methods detected pathogenic strains from apple orchards. Pss might exist in an apple tree during ice injury, and it secretes a toxin that makes leaves yellow and cause canker symptoms. Until now, Korea has not developed antibiotics targeting Pss. Therefore, it is necessary to develop effective disease control to combat Pss in apple orchards. Pathogenicity test on apple leaves and stems showed canker symptoms. The pathogenic bacterium was re-isolated from symptomatic plant tissue and confirmed as original isolates by 16S rRNA. Repetitive element sequence-based PCR and enterobacterial repetitive intergenic consensus PCR primers revealed different genetic profiles within P. syringae pathovars. High antibiotic susceptibility results showed the misreading of mRNA caused by streptomycin and oxytetracycline.

Aeromonas hydrophila ST251 and Aeromonas dhakensis are major emerging pathogens of striped catfish in Vietnam.

Aeromonads are ubiquitous in aquatic environments and several species are opportunistic pathogens of fish. Disease losses caused by motile Aeromonas species, particularly Aeromonas hydrophila, can be challenging in intensive aquaculture, such as at striped catfish (Pangasianodon hypophthalmus) farms in Vietnam. Outbreaks require antibiotic treatments, but their application is undesirable due to risks posed by resistance. Vaccines are an attractive prophylactic and they must protect against the prevalent strains responsible for ongoing outbreaks. This present study aimed to characterize A. hydrophila strains associated with mortalities in striped catfish culture in the Mekong Delta by a polyphasic genotyping approach, with a view to developing more effective vaccines. During 2013-2019, 345 presumptive Aeromonas spp. isolates were collected at farms in eight provinces. Repetitive element sequence-based PCR, multi-locus sequence typing and whole-genome sequencing revealed most of the suspected 202 A. hydrophila isolates to belong to ST656 (n = 151), which corresponds to the closely-related species Aeromonas dhakensis, with a lesser proportion belonging to ST251 (n = 51), a hypervirulent lineage (vAh) of A. hydrophila already causing concern in global aquaculture. The A. dhakensis ST656 and vAh ST251 isolates from outbreaks possessed unique gene sets compared to published A. dhakensis and vAh ST251 genomes, including antibiotic-resistance genes. The sharing of resistance determinants to sulphonamides (sul1) and trimethoprim (dfrA1) suggests similar selection pressures acting on A. dhakensis ST656 and vAh ST251 lineages. The earliest isolate (a vAh ST251 from 2013) lacked most resistance genes, suggesting relatively recent acquisition and selection, and this underscores the need to reduce antibiotics use where possible to prolong their effectiveness. A novel PCR assay was designed and validated to distinguish A. dhakensis and vAh ST251 strains. This present study highlights for the first time A. dhakensis, a zoonotic species that can cause fatal human infection, to be an emerging pathogen in aquaculture in Vietnam, with widespread distribution in recent outbreaks of motile Aeromonas septicaemia in striped catfish. It also confirms vAh ST251 to have been present in the Mekong Delta since at least 2013. Appropriate isolates of A. dhakensis and vAh should be included in vaccines to prevent outbreaks and reduce the threat posed by antibiotic resistance.

Molecular Characterization of Enterococcus faecalis and Enterococcus faecium Isolated from a Meat Source in Shahrekord Local Markets, Iran.

Enterococcus faecalis (E. faecalis) and E. faecium (E. faecium) are commensals of the gastrointestinal biota of humans and animals and are considered opportunistic pathogens. This study aimed to improve the knowledge about E. faecalis and E. faecium isolated from meat. To this end, 104 meat samples were collected from sheep, goats, cattle, and calves from Shahrekord local markets in Iran. Presumptive colonies of E. faecalis and E. faecium were isolated from the samples and subjected to identification tests. Antimicrobial susceptibility was determined using the Kirby-Bauer disc diffusion method, and polymerase chain reaction (PCR) was performed to detect some virulence genes. Furthermore, randomly amplified polymorphic DNA typing and repetitive element sequence-based PCR fingerprinting were used to show the clonal relatedness of the isolates. The results revealed that enterococci were present in 90 out of 104 samples. Specifically, E. faecalis and E. faecium were the commonly isolated species, with the predominance of E. faecalis, which exhibited high resistance to streptomycin (95%) but was susceptible to vancomycin (85.6%). Virulence genes detection showed that ccf and cpd genes were the most prevalent genes in both species. In addition, the molecular typing method indicated that the isolates belonged to separate subgroups. This study shows the contamination of meat products by potential pathogens and resistant enterococci. There is a need to implement regular surveillance to monitor the emergence of antimicrobial-resistant E. faecalis and E. faeciumin food, particularly in meat production.

Screening and risk assessment of biological control agents against bacterial wilt of ginger

Bacterial wilt caused by Ralstonia pseudosolanacearum race 4 is a highly destructive disease in ginger. To screen for biological control agents of the disease, 118 bacteria were isolated from different cultivating soils. First, the suppressive activity of soil bacteria on lesion development by R. pseudosolanacearum was evaluated using ginger leaves. Subsequently, a tobacco hypersensitive reaction test was performed for risk assessment in plants. After the clonality test by repetitive element sequence-based PCR, 12 screened isolates were inoculated into silkworm larvae as a preliminary risk assessment in animals, and we observed that five isolates did not cause a lethal effect. Finally, based on the results of the disease suppression test in infested soil, Pseudomonas sp. 008 was selected as the most suitable isolate for biological control. To ascertain the taxonomic position of the isolate, biochemical and physiological tests, multilocus sequence analysis, and average nucleotide identity with related species analysis were performed. Our results suggest that Pseudomonas sp. 008 is a novel species belonging to the P. mandelii subgroup of the P. fluorescens group. Risk assessment and taxonomic approaches with robust resolution demonstrated in the present study are expected to contribute to the efficient screening of biological control agents.

Identification, Genetic Diversity, and Chemical Control of Xanthomonas arboricola pv. pruni in China.

Peach bacterial spot caused by Xanthomonas arboricola pv. pruni has become widespread in most peach-producing areas of China and has caused devastating losses to the peach industry. However, little is known about the population biology and epidemiology of X. arboricola pv. pruni in China, thus no effective management strategy is available. Altogether, 321 symptomatic samples of peach bacterial spot from 12 provinces in China were collected from which 612 bacterial isolates were obtained. Based on 16S rDNA sequence comparison in GenBank, the obtained isolates were identified as Pantoea spp. (514) and Xanthomonas spp. (98). The pathogenicity test demonstrated that the causal agent of the peach bacterial spot was the Xanthomonas spp. instead of the Pantoea spp. Based on morphological observation, physiological and biochemical characterization, and molecular identification, the Xanthomonas spp. were further identified to be X. arboricola pv. pruni. Then, 41 X. arboricola pv. pruni isolates representing different populations were selected and analyzed with repetitive element sequence based-PCR and intersimple sequence repeat markers to understand the genetic diversity and population structure along with four X. arboricola pv. pruni isolates from plum and three isolates of X. arboricola pv. juglandis as comparison. A total of 98 polymorphic alleles were identified, with a mean value of percentage of polymorphic loci of 14. Genetic diversity and phylogenetic analysis revealed the profound heterogeneity between X. arboricola pv. juglandis and X. arboricola pv. pruni, moderate genetic differentiation within X. arboricola pv. pruni, and obvious host specificity but weak geographical differentiation in X. arboricola population. Finally, the efficiency of bactericides on X. arboricola pv. pruni was evaluated invitro and invivo. The parallel repeated field trials in two orchards demonstrated that 80% Mancozeb (1:800) and 47% Kocide (1:800, 1:1,500, and 1:2,000) had excellent control efficacies for X. arboricola pv. pruni, especially as the control efficacy of Kocide could even reach 90%. This study conducted a systematic investigation for the occurrence, population variance, and chemical control of X. arboricola pv. pruni. It improved the understanding of the pathogen populations of peach bacterial spot in China and provided solid theoretical and practical guidance for X. arboricola pv. pruni control.

Phenotypicand Genotypic Characterization of Clinical Isolates of Intracellular Adherent-Invasive Escherichia coli Among Different Stages, Family History, and Treated Colorectal Cancer Patients in Iran.

There is increasing evidence showing that microbial dysbiosis impacts the health and cancer risk of the host. An association between adherent–invasive Escherichia coli (AIEC) and colorectal cancer (CRC) has been revealed. Cyclomodulins (CMs) have been receiving increasing attention for carcinogenic changes. In this study, the incidence and features of intracellular AIEC and cyclomodulin-encoding genes were investigated and the phylogenetic grouping and genetic relatedness were evaluated. E. coli strains were isolated from the colorectal biopsies. Adhesion and invasion assays and intramacrophage cell survival test were performed to separate the AIEC isolates. Virulence genotyping for the genes htrA, dsbA, chuA, and lpfA and the cyclomodulin toxins was also conducted. In addition, phylogenetic grouping of the isolates was determined. Subsequently, repetitive element sequence-based PCR (rep-PCR) fingerprinting was performed. A total of 24 AIEC pathovars were isolated from 150 patients. The prevalence rates of htr, dsbA, and lpfA were 70.83% and that of chuA was 91.66%. The frequencies of the cyclomodulin toxins were as follows: cnf1, 29.2%; cnf2, 25%; colibactin, 29.2%; and cdt, 4.2%; cif was not found. Among the AIEC isolates, 4.2%, 4.2%, 54.2%, 29.2%, and 8.3% with phylotypes A or C, B1, B2, D, and E were identified, respectively. Left-sided colon carcinoma and adenocarcinoma T≥1 stage (CRC2) were colonized by B2 phylogroup AIEC-producing CMs more often than the samples from the other groups. Close genetic relatedness was observed in AIEC isolates with rep-PCR.

Identification and Genetic Characterization of Pseudomonas syringae pv.syringae from Sweet Cherry in Turkey.

Pseudomonas syringae pv. syringae, which causes bacterial canker, is the most polyphagous bacterium in the P. syringae complex because of its broad host range. This pathogen is considered the major bacterial disease in cherry orchards. In this study, several samples were collected from infected sweet cherry (Prunus avium L.) trees in different locations of the Marmara region in Turkey between 2016 and 2018. Sixty-three isolates were identified as P. syringae pv. syringae by pathogenicity, LOPAT, GATTa, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry tests. Total genomic DNA was extracted to confirm identity, followed by PCR amplification of syrB and cfl genes. Out of 63 isolates, 12 were randomly selected for repetitive element sequence-based PCR and multilocus sequence typing analyses to gain insight into the relationships of those isolates. The cluster analysis of enterobacterial repetitive intergenic consensus-, repetitive extragenic palindromic-, and BOX-A1R-based repetitive extragenic-palindromic-PCR techniques could classify the isolates into two distinct clusters. Phylogenetic analysis was carried out to obtain the relation between isolates and the location. The multilocus sequencing typing analysis of gyrB, rpoDp, rpoDs, and gltA genes allowed a clear allocation of the isolates into two separate main clusters. The relationships among the isolates were also evaluated by constructing a genealogical median-joining network (MJN). The isolates from six locations produced 11 haplotypes that were illustrated in the MJN. The results of this study proved that location could not be an indicator for showing the genetic diversity of P. syringae pv. syringae from cherry orchards. As the genetic variability of Pseudomonads has been demonstrated, this study also showed high diversity among different isolates even within the populations. While more research is recommended, the results of this study contributed to a better understanding of the evolutionary progress of P. syringae pv. syringae and the genetic diversity of sweet cherry isolates.

Phenotypic and Genotypic Characterization of Salmonella Isolates Recovered from Foods Linked to Human Salmonellosis Outbreaks in Minas Gerais State, Brazil

Salmonella is one of the primary pathogens that causes foodborne diseases worldwide. In the present study, we characterized Salmonella isolates recovered from foods linked to human salmonellosis outbreaks in Minas Gerais, Brazil, from 2003 to 2017. Serotype, antimicrobial susceptibility, presence of virulence genes, and genetic polymorphism as determined by repetitive element sequence-based PCR were determined for 70 Salmonella isolates. Thirteen Salmonella serotypes were identified, and the most prevalent were Enteritidis and Typhimurium, comprising 52 (74.3%) of the 70 isolates. Sixty-five (92.8%) of the isolates were resistant to at least 1 of the 15 antimicrobials tested. Ten isolates (14.2%) had a multidrug resistance phenotype. Isolates were screened for 16 virulence genes, which were found in 75.7 to 100% of the isolates. A statistical difference was found among Salmonella serotypes in the presence of the sipB, sopE, lfpA, sefA, and spvC genes. Based on their DNA fingerprints, 40 isolates of Salmonella Enteritidis from 16 outbreaks were separated into 14 groups and 12 isolates of Salmonella Typhimurium were separated into 6 groups. These serological patterns were similar to those reported by public health centers worldwide. Of concern is the high prevalence among the isolates in this study of both virulence genes and resistance to antimicrobials, especially to critically important drugs. Special attention should be given to Salmonella Enteritidis. Although the genomes of these Salmonella isolates were relatively variable, high genetic similarity was observed among them, and some had identical fingerprints. These results support the hypothesis of clonal circulation of Salmonella isolates causing human infections in Minas Gerais.

Identification and detection of pathogenic bacteria from patients with hospital-acquired pneumonia in southwestern Iran; evaluation of biofilm production and molecular typing of bacterial isolates

BackgroundHospital-acquired pneumonia (HAP) is the second most common nosocomial infection in intensive care units (ICUs). The present study aims to determine the prevalence of pathogenic bacteria, their biofilm formation, and molecular typing from patients with HAP in southwestern Iran.MethodsFifty-eight patients with HAP participated in this cross-sectional study. Sputum and endotracheal aspirate were collected from each patient for isolation and detection of bacteria. Biofilm formation was evaluated using Congo red agar or Microtiter plate assay. The antimicrobial susceptibility patterns of the isolates were investigated. The multiplex polymerase chain reaction (M-PCR) technique was used to determine the Staphylococcal Cassette Chromosome mec (SCCmec) types of methicillin-resistant Staphylococcus aureus (MRSA) strains. All S. aureus isolates were typed using the agr typing method. A repetitive element sequence-based PCR (rep-PCR) typing method was used for typing of Gram-negative bacteria. Data were analyzed using the Statistical Package for the Social Sciences (SPSS) software version 15 and the chi-square test.ResultsBacteria were isolated in 52 (89.7%) of patients. Acinetobacter baumannii (A. baumannii) was the most prevalent organism (37%), followed by S. aureus, Pseudomonas aeruginosa (P. aeruginosa), and Escherichia coli (E. coli). Using the PCR method, 56 bacteria were detected. A. baumannii was the most prevalent (35.7%) organism. A. baumannii and P. aeruginosa were biofilm-producing. All Gram-negative isolates were colistin-sensitive, and most of the A. baumannii isolates were multidrug-resistant (MDR). MRSA was identified in 12 (80%) S. aureus isolates, and 91.6% of MRSA were SCCmec type III. The agr type III was the most predominant. The rep-PCR analysis showed seven different patterns in 20 A. baumannii, six patterns in 13 P. aeruginosa, and four patterns in 6 E. coli.ConclusionA. baumannii was more prevalent than S. aureus in ventilator-associated pneumonia (VAP), while S. aureus is a major pathogen in non-ventilator hospital-acquired pneumonia (NV-HAP), possibly due to the tendency of the former to aquatic environments. Based on the rep-PCR typing method, it was concluded that bacteria were transmitted from patients or healthcare workers among different wards. Colistin can be used as a treatment in Gram-negative MDR isolates.

Antibiotic resistance, virulence-associated genes analysis and molecular typing of Klebsiella pneumoniae strains recovered from clinical samples

Klebsiella pneumoniae is a multidrug-resistant (MDR) opportunistic pathogen that causes nosocomial infections. Virulence analysis and molecular typing as powerful approaches can provide relevant information on K. pneumoniae infection. In the current study, antibiotic resistance, virulence-associated genes analysis, as well as molecular typing of K. pneumoniae strains were investigated. Out of 505 clinical samples collected from hospitalized patients, 100 K. pneumoniae strains were isolated by standard microbiological methods and subjected to the phenotypic and genotyping analysis. The highest prevalence of resistance was observed against ciprofloxacin (75%), trimethoprim–sulfamethoxazole (73%) and nitrofurantoin (68%). Virulence associated genes including entB, traT, ybts, magA, iucC, htrA and rmpA were found in 80%, 62%, 75%, 5%, 30%, 72% and 48%, of the isolates, respectively. The prevalence of biofilm-associated genes including mrkA, fimH, and mrkD were equally 88% for all tested isolates. Moreover, the efflux pump genes including AcrAB, TolC and mdtK were observed in 41 (41%), 33 (33%) and 26 (26%) of the strains respectively. A significant statistical association was observed between MDR strains and high expression of efflux pump and biofilm genes. The K. pneumoniae strains were differentiated into 11 different genetic patterns using the repetitive element sequence-based PCR (rep-PCR) technique. High prevalence of resistance, presence of various virulence factors, high level of efflux pump, and biofilm gene expression in diverse clones of K. pneumoniae strains pose an important health issue in clinical settings.

Classification of a Violacein-Producing Psychrophilic Group of Isolates Associated with Freshwater in Antarctica and Description of Rugamonas violacea sp. nov.

ABSTRACTA group of 11 bacterial strains was isolated from streams and lakes located in a deglaciated northern part of James Ross Island, Antarctica. They were rod-shaped, Gram-stain-negative, motile, and catalase-positive and produced blue-violet-pigmented colonies on R2A agar. A polyphasic taxonomic approach based on 16S rRNA gene sequencing, whole-genome sequencing, automated ribotyping, repetitive element sequence-based PCR (rep-PCR), MALDI-TOF MS, fatty acid profile, chemotaxonomy analyses, and extensive biotyping was applied in order to clarify the taxonomic position of these isolates. Phylogenetic analysis based on the 16S rRNA gene indicated that all the isolates constituted a coherent group belonging to the genus Rugamonas. The closest relatives to the representative isolate P5900T were Rugamonas rubra CCM 3730T, Rugamonas rivuli FT103WT, and Rugamonas aquatica FT29WT, exhibiting 99.2%, 99.1%, and 98.6% 16S rRNA pairwise similarity, respectively. The average nucleotide identity and digital DNA-DNA hybridization values calculated from the whole-genome sequencing data clearly proved that P5900T represents a distinct Rugamonas species. The G+C content of genomic DNAs was 66.1 mol%. The major components in fatty acid profiles were summed feature 3 (C16:1 ω7c/C16:1 ω6c), C 16:0, and C12:0. The cellular quinone content contained exclusively ubiquinone Q-8. The predominant polar lipids were diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine. The polyamine pattern was composed of putrescine, 2-hydroxputrescine, and spermidine.IMPORTANCE Our polyphasic approach provides a new understanding of the taxonomy of novel pigmented Rugamonas species isolated from freshwater samples in Antarctica. The isolates showed considerable extracellular bactericidal secretions. The antagonistic activity of studied isolates against selected pathogens was proved by this study and implied the importance of such compounds’ production among aquatic bacteria. The psychrophilic and violacein-producing species Roseomonas violacea may play a role in the diverse consortium among pigmented bacteria in the Antarctic water environment. Based on all the obtained results, we propose a novel species for which the name Rugamonas violacea sp. nov. is suggested, with the type strain P5900T (CCM 8940T; LMG 32105T). Isolates of R. violacea were obtained from different aquatic localities, and they represent the autochthonous part of the water microbiome in Antarctica.

Pandora's box in the dental clinic.

In June 2018, the Ministry of Health received notification from 2 hospitals about 2 patients who presented with overwhelming Enterobacter kobei sepsis that developed within 24 hours after a dental procedure. We describe the investigation of this outbreak. The epidemiologic investigation included site visits in 2 dental clinics and interviews with all involved healthcare workers. Chart reviews were conducted for case and control subjects. Samples were taken from medications and antiseptics, environmental surfaces, dental water systems, and from the involved healthcare professionals. Isolate similarity was assessed using repetitive element sequence-based polymerase chain reaction (REP-PCR). The 2 procedures were conducted in different dental clinics by different surgeons and dental technicians. A single anesthesiologist administered the systemic anesthetic in both cases. Cultures from medications, fluids and healthcare workers' hands were negative, but E. kobei was detected from the anesthesiologist's portable medication cart. The 2 human isolates and the environmental isolate shared the same REP-PCR fingerprinting profile. None of the 21 patients treated by the anesthesiologist in a general hospital during the same period, using the hospital's medications, developed infection following surgery. An outbreak of post-dental-procedure sepsis was linked to a contaminated medication cart, emphasizing the importance of medication storage standards and strict aseptic technique when preparing intravenous drugs during anesthesia. Immediate reporting of sepsis following these outpatient procedures enabled early identification and termination of the outbreak.

Characterization of Salmonella Isolates from Various Geographical Regions of the Caucasus and Their Susceptibility to Bacteriophages

Non-typhoidal Salmonella present a major threat to animal and human health as food-borne infectious agents. We characterized 91 bacterial isolates from Armenia and Georgia in detail, using a suite of assays including conventional microbiological methods, determining antimicrobial susceptibility profiles, matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry, serotyping (using the White-Kauffmann-Le Minor scheme) and genotyping (repetitive element sequence-based PCR (rep-PCR)). No less than 61.5% of the isolates were shown to be multidrug-resistant. A new antimicrobial treatment strategy is urgently needed. Phage therapy, the therapeutic use of (bacterio-) phages, the bacterial viruses, to treat bacterial infections, is increasingly put forward as an additional tool for combatting antibiotic resistant infections. Therefore, we used this representative set of well-characterized Salmonella isolates to analyze the therapeutic potential of eleven single phages and selected phage cocktails from the bacteriophage collection of the Eliava Institute (Georgia). All isolates were shown to be susceptible to at least one of the tested phage clones or their combinations. In addition, genome sequencing of these phages revealed them as members of existing phage genera (Felixounavirus, Seunavirus, Viunavirus and Tequintavirus) and did not show genome-based counter indications towards their applicability against non-typhoidal Salmonella in a phage therapy or in an agro-food setting.

Genetic diversity and molecular epidemiology of outbreaks of Klebsiella pneumoniae mastitis on two large Chinese dairy farms

Klebsiella pneumoniae is an opportunistic and environmental mastitis-causing pathogen, with potential for contagious transmission. Repetitive element sequence-based PCR was used to determine genetic diversity and explore potential transmission and reservoirs for mastitis caused by K. pneumoniae on 2 large Chinese dairy farms. A total of 1,354 samples was collected from the 2 dairy farms, including milk samples from cows with subclinical and clinical mastitis, bedding, feces, feed, teat skin, and milking liners. Environmental samples were collected from all barns and milking parlors and extramammary samples from randomly selected dairy cows on both farms. In total, 272 and 93 K. pneumoniae isolates were obtained from Farms A and B, respectively (with ~8K and 2K lactating cows, respectively). Isolation rates from clinical mastitis (CM), subclinical mastitis (SCM), and environmental or extramammary samples were 34, 23 and 37%, respectively for Farm A and 42, 3, and 34% for Farm B. The K. pneumoniae isolated from CM milk and extramammary or environmental sources had high genetic diversity (index of diversity >90%) on the 2 farms and from SCM on Farm A. However, on Farm B, 9 SCM isolates were classified as 2 genotypes, resulting in a relatively low index of diversity (Simpson's index of diversity = 0.39; 95% CI = 0.08-0.70). Genotypes of K. pneumoniae causing mastitis were commonly detected in feces, bedding, and milking liners (Farm A), or from teat skin, sawdust bedding, and feed (Farm B). Based on its high level of genetic diversity, we inferred K. pneumoniae was an opportunistic and environmental pathogen causing outbreaks of CM on these 2 large Chinese dairy farms. Nevertheless, that only a few genotypes caused SCM implied some strains had increased udder adaptability and a contagious nature or a common extramammary source. Finally, control of intramammary infections caused by K. pneumoniae on large Chinese dairy farms must consider farm-level predictors, as the 2 outbreaks had distinct potential environmental sources of infection.

A survey for phylogenetic relationship; presence of virulence genes and antibiotic resistance patterns of avian pathogenic and uropathogenic Escherichia coli isolated from poultry and humans in Yazd, Iran

IntroductionExtra-intestinal infections in humans and poultry are caused by Extra-intestinal Pathogenic Escherichia coli strains (ExPEC) such as Avian Pathogenic E. coli (APEC) and Uropathogenic E. coli (UPEC). This study aimed to compare the presence of four pathogenic genes and antimicrobial resistance patterns of APEC and UPEC isolates, as well as investigating the phylogenetic relationship between these isolates by the Repetitive element sequence-based PCR (rep-PCR) method. Materials and methods112 E. coli isolates were collected from poultry specimens in farms and 88 E. coli isolates from human urinary tract infection in the patients of the hospital in Yazd city during March 2015 to July 2016. Susceptibility testing was carried out using the disk diffusion method. PCR was performed to investigate the presence of fimH, iucC, sitA, and irp2 genes. Data were analyzed using SPSS software (version 16) and Chi-square test. Phylogenetic analysis was performed and analyzed by gel-comparison software for the rep-PCR method. ResultsThe frequency of fimH, iucC, sitA, and irp2 genes was 67.8, 78.5, 84.8 and 89.2% for APEC and 96.5, 79.5, 86.3 and 75% for UPEC, respectively. Poultry strains exhibited multi-drug resistance to 16 antibiotics with the highest resistance to nalidixic acid and erythromycin, and all human isolates showed multi-drug resistance. The phylogenetic relationship was not found between human and poultry isolates by the rep-PCR method. ConclusionHigh antibiotic resistance was observed in isolates related to humans and poultry. Due to the presence of fimH, iucC, sitA, and irp2 genes in APEC and UPEC isolates, these genes are suggested to be effective in extra-intestinal E. coli infections. The phylogenetic relationship between the two pathotypes was not found by rep-PCR. Therefore, the hypothesis of transferring APEC to humans requires further investigation.

Differential Genotyping of Mycobacterium avium Complex and Its Implications in Clinical and Environmental Epidemiology.

In recent decades, the incidence and prevalence of nontuberculous mycobacteria (NTM) have greatly increased, becoming a major worldwide public health problem. Among numerous NTM species, the Mycobacterium avium complex (MAC) is the most predominant species, causing disease in humans. MAC is recognized as a ubiquitous microorganism, with contaminated water and soil being established sources of infection. However, the reason for the recent increase in MAC-associated disease has not yet been fully elucidated. Furthermore, human MAC infections are associated with a variety of infection sources. To improve the determination of infection sources and epidemiology of MAC, feasible and reliable genotyping methods are required to allow for the characterization of the epidemiology and biology of MAC. In this review, we discuss genotyping methods, such as pulsed-field gel electrophoresis, a variable number of tandem repeats, mycobacterial interspersed repetitive-unit-variable number of tandem repeats, and repetitive element sequence-based PCR that have been applied to elucidate the association between the MAC genotypes and epidemiological dominance, clinical phenotypes, evolutionary process, and control measures of infection. Characterizing the association between infection sources and the epidemiology of MAC will allow for the development of novel preventive strategies for the effective control of MAC infection.

Correct species identification (reclassification in CNCTC) of strains of Staphylococcus intermedius-group can improve an insight into their evolutionary history.

A group of 59 putative strains of Staphylococcus intermedius/Staphylococcus pseudintermedius deposited in the Czech National Collection of Type Cultures (CNCTC, National Institute for Public Health, Prague, Czech Republic) and the National Reference Laboratory for Staphylococci (NRL for Staphylococci, National Institute for Public Health, Prague, Czech Republic) was reclassified using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). There the biggest human collection of S. pseudintermedius in Europe was analysed; 44 samples (75%) were of human origin. Twenty-two percent (n= 13) of the strains were isolated from animals, and two staphylococci were of unknown origin. This study revealed the prevalence of Staphylococcus pseudintermedius (94%, n= 53) vs. Staphylococcus intermedius (6%, n= 6) in the collection of human and veterinary staphylococci after reclassification. Results of PCR-RFLP analysis were verified by comparison with a repetitive element sequence-based polymerase chain reaction (Rep-PCR) analysis on 26 (44%) randomly selected strains. Due to a low-resolution ability of PCR-RFLP to separate Staphylococcus intermedius from Staphylococcus delphini, four isolates of Staphylococcus intermedius were biochemically verified further to exclude the presence of Staphylococcus delphini in the collection. Our results indicate that S. intermedius and S. pseudintermedius have occurred independently over an age-long period of their co-evolution.

Detection of Salmonella in the intestine of Hypostomus plecostomus from the upper San Marcos River, Texas.

The prevalence of salmonellae in the intestines of the invasive suckermouth catfish Hypostomus plecostomus was assessed in the San Marcos River, just down-stream of its spring-fed headwaters. In 2014, H. plecostomus, sediment, and water samples were collected during 15 sampling events. A combination of semi-selective enrichment and quantitative polymerase chain reaction (qPCR) revealed the presence of salmonellae in 45% of the fish intestines across the entire year, with a prevalence range of 13-100% per sampling event. Repetitive element sequence-based PCR (rep-PCR) and multi-locus sequence typing (MLST) revealed a high diversity of salmonellae from fish intestine samples at individual sampling times, single or multiple presence of rep-PCR patterns and serotypes within individual fish, and identical rep-PCR patterns and serotypes for different fish within and across sampling events. Overall, 15 serotypes were identified by MLST, with a diversity range between one and seven serotypes per sampling event. Some serotypes were retrieved only once, while others were detected more frequently. A few serotypes were retrieved at several sampling times, nearly evenly distributed over the entire sampling period. Prevalence and diversity were independent of precipitation events, indicating the potential presence of environmental strains that are capable of long-term persistence in the environment.

Clinical isolates of Escherichia coli solely resistant to mecillinam: prevalence and epidemiology

In routine susceptibility testing of Gram-negative bacteria, a particular resistance phenotype was observed: an Escherichia coli isolate from a urine sample exhibited resistance solely to mecillinam (MEC) but was fully susceptible to other β-lactam antibiotics (MEC-R-BL-S). The objectives as this study were to determine the prevalence of this phenotype and to describe the phenotype, molecular epidemiology and genetic background. Between 1 January 2014 and 31 January 2016, MEC-R-BL-S E. coli isolates from urine were collected and genes previously reported as mostly involved in MEC resistance were analysed. The genetic relatedness among isolates was investigated by repetitive element sequence-based PCR (rep-PCR) and multilocus sequence typing (MLST). Ten MEC-R-BL-S isolates were collected, accounting for 0.4% (10/2547) of all E. coli obtained from urine samples, 0.9% (10/1135) of ampicillin-susceptible E. coli isolates and 9.6% (10/104) of MEC-R E. coli isolates. The isolates appeared as small colonies with round morphology and had impaired fitness. The isolates were not clonal and belonged to various extraintestinal or commensal E. coli phylogroups. Mutations in the cysB gene were evidenced in all clinical isolates. In conclusion, microbiologists should be aware of these isolates with a particular susceptibility phenotype, which is not due to error in disk diffusion but is a real non-enzymatic antibiotic resistance pattern.