Repetitive Sequence-based PCR Research Articles

Multidrug-Resistant Enteropathogenic Escherichia coli Isolated from Diarrhoeic Calves, Milk, and Workers in Dairy Farms: A Potential Public Health Risk.

Enteropathogenic Escherichia coli (EPEC) is a leading cause of diarrhoeagenic diseases in humans and cattle worldwide. The emergence of multidrug-resistant (MDR) EPEC from cattle sources is a public health concern. A total of 240 samples (75 diarrhoeic calves, 150 milk samples, and 15 workers) were examined for prevalence of EPEC in three dairy farms in Egypt. Antimicrobial resistance (AMR) traits were determined by antibiogram and polymerase chain reaction (PCR) detection of β-lactamase-encoding genes, plasmid-mediated quinolone resistance genes, and carbapenemase-encoding genes. The genetic relatedness of the isolates was assessed using repetitive extragenic palindromic sequence-based PCR (REP-PCR). EPEC isolates were detected in 22.7% (17/75) of diarrhoeic calves, 5.3% (8/150) of milk samples, and 20% (3/15) of worker samples. The detected serovars were O26 (5%), O111 (3.3%), O124 (1.6%), O126 (0.8%), and O55 (0.8%). AMR-EPEC (harbouring any AMR gene) was detected in 9.2% of samples. Among isolates, blaTEM was the most detected gene (39.3%), followed by blaSHV (32.1%) and blaCTX-M-1 (25%). The qnrA, qnrB, and qnrS genes were detected in 21.4%, 10.7%, and 7.1% of isolates, respectively. The blaVIM gene was detected in 14.3% of isolates. All EPEC (100%) isolates were MDR. High resistance rates were reported for ampicillin (100%), tetracycline (89.3%), cefazolin (71%), and ciprofloxacin (64.3%). Three O26 isolates and two O111 isolates showed the highest multiple-antibiotic resistance (MAR) indices (0.85–0.92); these isolates harboured blaSHV-12 and blaCTX-M-15 genes, respectively. REP-PCR genotyping showed high genetic diversity of EPEC, although isolates belonging to the same serotype or farm were clustered together. Two worker isolates (O111 and O26) showed high genetic similarity (80–95%) with diarrhoeic calf isolates of matched serotypes/farms. This may highlight potential inter-species transmission within the farm. This study highlights the potential high risk of cattle (especially diarrhoeic calves) as disseminators of MDR-EPEC and/or their AMR genes in the study area. Prohibition of non-prescribed use of antibiotics in dairy farms in Egypt is strongly warranted.

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.

Comparison of Whole Genome Sequencing and Repetitive Element PCR for Multidrug-Resistant Pseudomonas aeruginosa Strain Typing

Hospital-acquired infections pose significant costly global challenges to patient care. Rapid and sensitive methods to identify potential outbreaks are integral to infection control measures. Whole-genome sequencing (WGS)-based bacterial strain typing provides higher discriminatory power over standard nucleotide banding pattern-based methods such as repetitive sequence-based PCR (rep-PCR). However, integration of WGS into clinical epidemiology is limited by the lack of consensus in methodology and data analysis/interpretation. In this study, WGS was performed on genomic DNA extracted from 22 multidrug-resistant Pseudomonas aeruginosa (MDR-PA) isolates using next-generation sequencing. Resulting high-quality reads were analyzed for phylogenetic relatedness using a whole-genome multilocus sequence typing (wgMLST)-based software program and single-nucleotide variant phylogenomics (SNVPhyl). WGS-based results were compared with conventional MLST and archived rep-PCR results. Rep-PCR identified three independent clonal clusters of MDR-PA. Only one clonal cluster identified by rep-PCR, an endemic strain within the pediatric cystic fibrosis population at Texas Children's Hospital, was concordantly identified using wgMLST and SNVPhyl. Results were highly consistent between the three sequence-based analyses (conventional MLST, wgMLST, and SNVPhyl), and these results remained consistent with the addition of 74 MDR-PA genomes. These WGS-based methods provided greater resolution for strain discrimination than rep-PCR or standard MLST classification, and the ease of use of wgMLST software renders it clinically viable for analysis, interpretation, and reporting of WGS-based strain typing.

Genetic Diversity and Population Structure of the Xanthomonas campestris pv. campestris Strains Affecting Cabbages in China Revealed by MLST and Rep-PCR Based Genotyping.

Xanthomonas campestris pv. campestris (Xcc) is the causal agent of black rot for cruciferous vegetables worldwide, especially for the cole crops such as cabbage and cauliflower. Due to the lack of resistant cabbage cultivars, black rot has brought about considerable yield losses in recent years in China. Understanding of the pathogen features is a key step for disease prevention, however, the pathogen diversity, population structure, and virulence are largely unknown. In this study, we studied 50 Xcc strains including 39 Xcc isolates collected from cabbage in 20 regions across China, using multilocus sequence genotyping (MLST), repetitive DNA sequence-based PCR (rep-PCR), and pathogenicity tests. For MLST analysis, a total of 12 allelic profiles (AP) were generated, among which the largest AP was AP1 containing 32 strains. Further cluster analysis of rep-PCR divided all strains into 14 DNA groups, with the largest group DNA I comprising of 34 strains, most of which also belonged to AP1. Inoculation tests showed that the representative Xcc strains collected from diverse regions performed differential virulence against three brassica hosts compared with races 1 and 4. Interestingly, these results indicated that AP1/DNA I was not only the main pathotype in China, but also a novel group that differed from the previously reported type races in both genotype and virulence. To our knowledge, this is the first extensive genetic diversity survey for Xcc strains in China, which provides evidence for cabbage resistance breeding and opens the gate for further cabbage-Xcc interaction studies.

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.

Antimicrobial resistance profiling and molecular typing of ruminant-borne isolates of Clostridium perfringens from Xinjiang, China

ObjectivesClostridium perfringens (C. perfringens) can cause intestinal diseases in livestock and humans, which seriously threatens the healthy development of animal husbandry and human food safety. Here, the characteristics of antimicrobial resistance and molecular typing of ruminant-borne strains of C. perfringens in Xinjiang, China were explored and profiled. MethodsA total of 307 clinical feces collected from ruminants (cattle and sheep) with diarrheal symptoms were screened for C. perfringens. The recovered isolates were characterized in respect to their antimicrobial resistance pattern and molecular typing. ResultsA total of 109 isolates of C. perfringens were isolated from 307 clinical feces of ruminants, most of which displayed the multidrug resistance (MDR) phenotype. Demonstration of the quinolone-resistance gene was the highest among the isolates (70.6%). The multiplex PCR typing based on toxin genes showed that type A and type D strains made up 82.6% (90/109) and 17.4% (19/109), among which, the isolates carrying β2 gene occupied 43.3% (39/90) of type A strains and 31.6% (6/19) of type D strains. These isolates were divided into 6 genotypes (I-VI) by enterobacterial repetitive intergenic consensus sequence-based PCR (ERIC-PCR) method. A total of 33 ST types (ST1-ST33) were identified by multilocus sequence typing (MLST) method. ConclusionC. perfringens isolates with multidrug resistance (MDR) were frequent and circulating in ruminants. Among them, type A-Ⅰ-ST19 was the dominant genotype of C. perfringens, displaying obvious genetic diversity. This study provided important epidemiological data for the risk assessment of food safety associated with ruminant-borne C. perfringens in Xinjiang, China.

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.

GTG5 fingerprinting of native Streptococcus thermophilus strains and its authentication by principal component analysis – A road to value added commercial yoghurt starter cultures

There is always interest in investigating new lactic acid bacteria strains for product development that can complement or replace currently-in-use starters. This study screened native strains of Streptococcus thermophilus from various plant/dairy sources across India and compared them with commercial yoghurt starters. A total of 158 isolates were characterised as S. thermophilus by polyphasic approach and strain typed using (GTG)5 primers by repetitive sequence-based polymerase chain reaction. After cluster analysis, 12 major clusters were defined at a similarity level of 70%. They were evaluated for functionally important metabolic traits and representative genotypes with desired starter activity were used for yoghurt production along with commercial starter. Based on physico-chemical attributes, sensory and principal component analysis, yoghurt prepared using representative plant and dairy isolates were indistinguishable from product prepared using reference yoghurt strains. The identified strains were promising with additional functional attributes such as galactose fermenting ability and negative for urease activity.

Genetic variability of Edwardsiella piscicida isolates from Mississippi catfish aquaculture with an assessment of virulence in channel and channel × blue hybrid catfish.

The bacterium Edwardsiella piscicida causes significant losses in global aquaculture, particularly channel (Ictalurus punctatus)×blue (I.furcatus) hybrid catfish cultured in the south-eastern United States. Emergence of E.piscicida in hybrid catfish is worrisome given current industry trends towards increased hybrid production. The project objectives were to assess intraspecific genetic variability of E.piscicida isolates recovered from diseased channel and hybrid catfish in Mississippi; and determine virulence associations among genetic variants. Repetitive extragenic palindromic sequence-based PCR (rep-PCR) using ERIC I and II primers was used to screen 158 E.piscicida diagnostic case isolates. A subsample of 39 E.piscicida isolates, representing predominant rep-PCR profiles, was further characterized using BOX and (GTG)5 rep-PCR primers, virulence gene assessment and multilocus sequence analysis (MLSA) targeting housekeeping genes gyrb, pgi and phoU. The MLSA provided greater resolution than rep-PCR, revealing 5 discrete phylogroups that correlated similarly with virulence gene profiles. Virulence assessments using E.piscicida representatives from each MLSA group resulted in 14-day cumulative mortality ranging from 22% to 54% and 63 to 72% in channel and hybrid fingerlings, respectively. Across all phylogroups, mortality was higher in hybrid catfish (p<.05), supporting previous work indicating E.piscicida is an emerging threat to hybrid catfish aquaculture in the south-eastern United States.

Insights from Bacteroides Species in Children with Type 1 Diabetes.

In our previous study the enrichment of the intestinal proteome of type 1 diabetes (T1D) children with Bacteroides proteins was observed, which led us to our current study that aimed to isolate and characterize Bacteroides species from fecal samples of T1D and control children. Repetitive sequence-based PCR (rep-PCR) was used for typing the isolated Bacteroides species. The antibiotic susceptibility and mucinolytic activity of the isolates was determined. The quantification of specific bacterial groups in the fecal samples was determined by qPCR. The ability to adhere and invade the human colonic cell line HT29-MTX-E12 of strains of P. dorei, B. uniformis and P. distasonis was determined and their whole genome sequencing was performed. The results showed similar numbers of Bacteroides species in T1D and control samples, but unique Bacteroides species and a higher recovery of P. distasonis from T1D samples was observed. Rep-PCR grouped the different Bacteroides species, but no discrimination by origin was achieved. T1D children showed a significant increase in Proteobacteria and a depletion in Lactobacillus sp. All tested P. dorei, B. uniformis and P. distasonis were able to adhere to HT29-MTX-E12 cells but significant differences (p < 0.05) in the ability to invade was observed. The highest ability to invade was exhibited by P. distasonis PtF D14MH1 and P. dorei PtFD16P1, while B. uniformis strains were unable to invade. The damage to tight junctions was also observed. The presence of Lactobacillus sp. inhibited the invasion ability of P. distasonis PtF D14MH1 but not P. dorei PtFD16P1. Sequences of agonist peptides of the human natural preproinsulin and the insulin B chain insB:9-23 peptide mimics were identified. The results reported in our study stresses the continued efforts required to clarify the link between T1D and gut microbiota.

From the Urinary Catheter to the Prevalence of Three Classes of Integrons, β-Lactamase Genes, and Differences in Antimicrobial Susceptibility of Proteus mirabilis and Clonal Relatedness with Rep-PCR.

Introduction Proteus mirabilis is a biofilm-forming agent that quickly settles on the urinary catheters and causing catheter-associated urinary tract infections. Thus, the spread of multidrug-resistant P. mirabilis isolates, with the ability to form a biofilm that carries integron, extended-spectrum β-lactamases (ESBLs), and plasmid-mediated colistin resistance genes (mcr), represents a severe threat to managing nosocomial infectious diseases. This study is aimed at surveying the prevalence of ESBL, integrase, and mcr genes of P. mirabilis, isolated from the catheter, to assess the differences in their antimicrobial susceptibility and clonal dissemination. Method Microtiter plate assay was adopted to measure biofilm formation. The antimicrobial susceptibility was assessed by the disk diffusion method. Antimicrobial resistance genes (intI1, intI2, intI3, blaTEM, blaCTX-M, blaSHV, mcr1, and mcr2) were detected by PCR. All of the isolates were characterized by repetitive sequence-based PCR. Result From 385 collected catheters in patients admitted to the intensive care unit (ICU), 40 P. mirabilis were isolated. All of the isolates could form a biofilm. Proteus spp. had intrinsic resistance to tetracycline (95%) and nitrofurantoin (92.5%), which explains the high resistance prevalence. The most widely resistant antibiotic was trimethoprim-sulfamethoxazole (75%). Thirty-three (82.5%) isolates were classified as multidrug resistance (MDR). The prevalence of intI1 and intI2 genes was 60% and 25%, respectively. In 6 (15%) isolates, both genes were detected. The most frequent ESBL gene detected in all of the isolates was blaTEM. Also, no detection for mcr1 and mcr2 antibiotic resistance genes was reported. Rep-PCR identified 39(GTG)5 types (G1–G39) of 40 isolates that 38 isolates had unique patterns. Conclusion In this study, 82.5% of isolates were MDR with high antibiotic resistance to trimethoprim-sulfamethoxazole. The intI1 and blaTEM were the most prevalent genes in the integrase and ESBL gene family. High diversity was seen in the isolates with Rep-PCR. The increasing rate of MDR isolates with a high prevalence of resistance genes could be alarming and demonstrate the need for hygienic procedures to prevent the increased antibiotic resistance rate in the future.

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.

Clonal Relationship within Two Oral Actinomyces Populations Collected from Plaque of Periodontitis Patients

Actinomyces naeslundii and A. oris are dental plaque formers involved in the pathogenesis of periodontitis. The aim of the study was to investigate the clonal relationship within two oral Actinomyces populations collected from plaque of patients with chronic periodontitis. The 223 clinical strains of A. naeslundii and A. oris were isolated from biofilm samples collected supra and subgingivally from teeth with shallow (probing depth (PD) = 3-4 mm), deep (PD = 5-6 mm) and very deep (PD ≥7 mm) pockets from 20 chronic periodontitis patients. All strains were submitted to repetitive sequence-based PCR typing using DiversiLab (BioMerieux,Marcy l´Étoile, France). Seven patients harboured only unrelated (&lt;95% similarity) multiple isolates, while 13 harboured both similar (&gt;95% similarity) and unrelated isolates at different sites. Identical (&gt;98% similarity) strains were found to be present in the subgingival shallow depths more often than in the other subgingival depths. The number of clones in individual patients varied from 2 to 17 different rep-PCR genotypes. The clonal relationship within the oral populations of A. naeslundii and A. oris in an individual was unpredictable, ranging from the presence of multiple genotypes with no clonal similarity to only two different clones supra or subgingivally at different sites.

Molecular Epidemiology of Carbapenem-Resistant Acinetobacter baumannii Isolates from Northern Africa and the Middle East.

At the Bundeswehr Hospitals of Hamburg and Westerstede, patients repatriated from subtropical war and crisis zones of Northern Africa and the Middle East were medically treated, including microbiological assessment. Within a six-year interval, 16 Acinetobacter spp. strains, including 14 Acinetobacter baumannii (Ab) isolates with resistance against carbapenems and origins in Afghanistan (n = 4), Iraq (n = 2), Libya (n = 2), and Syria (n = 8) were collected. While clonal relationships of Libyan and Syrian strains had been assessed by superficial next generation sequencing (NGS) and “DiversiLab” repetitive elements sequence-based (rep-)PCR so far, this study provides core genome-based sequence typing and thus more detailed epidemiological information. In detail, sequencing allowed a definitive species identification and comparison with international outbreak-associated Ab strains by core genome multi locus sequence typing (cgMLST) and the identification of MLST lineages, as well as the identification of known resistance genes. The sequence analysis allowed for the confirmation of outbreak-associated clonal clusters among the Syrian and Afghan Ab isolates, indicating likely transmission events. The identified acquired carbapenem resistance genes comprised blaOXA-23, blaOXA-58, blaNDM-1, and blaGES-11, next to other intrinsic and acquired, partly mobile resistance-associated genes. Eleven out of 14 Ab isolates clustered with the previously described international clonal lineages IC1 (4 Afghan strains), IC2 (6 Syrian strains), and IC7 (1 Syrian strain). Identified Pasteur sequence types of the 14 Ab strains comprised ST2 (Syrian), ST25 (Libyan), ST32 (Iraqi), ST81 (Afghan), ST85 (Libyan), and ST1112 (Syrian), respectively. In conclusion, the study revealed a broad spectrum of resistance genes in Ab isolated from war-injured patients from Northern Africa and the Middle East, thereby broadening the scarcely available data on locally abundant clonal lineages and resistance mechanisms.

Advanced Killing Potential of Thymol against a Time and Temperature Optimized Attached Listeria monocytogenes Population in Lettuce Broth.

Fresh vegetables and salads are increasingly implicated in outbreaks of foodborne infections, such as those caused by Listeria monocytogenes, a dangerous pathogen that can attach to the surfaces of the equipment creating robust biofilms withstanding the killing action of disinfectants. In this study, the antimicrobial efficiency of a natural plant terpenoid (thymol) was evaluated against a sessile population of a multi-strain L. monocytogenes cocktail developed on stainless steel surfaces incubated in lettuce broth, under optimized time and temperature conditions (54 h at 30.6 °C) as those were determined following response surface modeling, and in comparison, to that of an industrial disinfectant (benzalkonium chloride). Prior to disinfection, the minimum bactericidal concentrations (MBCs) of each compound were determined against the planktonic cells of each strain. The results revealed the advanced killing potential of thymol, with a concentration of 625 ppm (= 4 × MBC) leading to almost undetectable viable bacteria (more than 4 logs reduction following a 15-min exposure). For the same degree of killing, benzalkonium chloride needed to be used at a concentration of at least 20 times more than its MBC (70 ppm). Discriminative repetitive sequence-based polymerase chain reaction (rep-PCR) also highlighted the strain variability in both biofilm formation and resistance. In sum, thymol was found to present an effective anti-listeria action under environmental conditions mimicking those encountered in the salad industry and deserves to be further explored to improve the safety of fresh produce.

Genetic diversity of potato blackleg pathogens, Pectobacterium wasabiae, P. carotovorum subsp. brasiliense and Dickeya dianthicola in Japan by rep-PCR fingerprinting

We determined the species composition of 219 isolates of potato blackleg or soft rot pathogens in Japan collected after 2000 from potato plants by species-specific PCR. Of the total, Pectobacterium wasabiae (Pw) and P. carotovorum subsp. brasiliense (Pcb) comprised 88% (Pw: 94 isolates, Pcb: 98 isolates) of the isolates and Dickeya dianthicola (Ddi) 12% (27 isolates). P. atrosepticum (Pa) was not detected. Genetic diversity of Pw, Pcb and Ddi was investigated using repetitive sequence-based PCR (rep-PCR) fingerprinting with three primer pairs complementary to repetitive sequences in the bacterial genome (BOX, ERIC and REP). One hundred and twenty-one Pw isolates, one hundred Pcb isolates and thirty-three Ddi isolates were assigned to twenty-nine, twenty-one and eleven fingerprint genotypes (FGs) based on the combination of fingerprint patterns for each rep-PCR experiment (BOX-, ERIC- and REP-PCR). The genetic diversity of Pw and Ddi isolates in Japan was considered high, with Simpson’s diversity index and Shannon’s index of 0.87 and 2.44 for Pw, and 0.84 and 2.05 for Ddi, respectively. Pcb isolates were less diversified genetically (diversity index: 0.57 and 1.58). The FGs of the isolates obtained from blackleg-affected potato plants in the seed tuber field agreed with those of the isolates in the field where the seeds produced were grown. These results support the occurrence of seed-borne transmission of the blackleg pathogen between the fields.

Colistin-Resistant Enterobacterales Isolated from Chicken Meat in Western Algeria.

Aim: In Algeria, colistin is used as a metaphylactic treatment in the poultry industry for the treatment of Gram-negative gastrointestinal infections and also as a feed additive to promote animal growth. The aim of this study was to investigate the importance and genetic characteristics of colistin-resistant Enterobacterales from chicken meat in Western Algeria. Results: A total of 181 samples of chicken meat were collected from three poultry farms across three provinces in Western Algeria. The presence of colistin-resistant Enterobacterales isolates was screened on selective media. Resistance and virulence profiles were characterised by PCR and sequencing. The clonal relatedness of the different mcr positive isolates was studied using repetitive sequence-based PCR (Rep-PCR) and multilocus sequence typing. Transferability and characteristics of plasmids harboring mcr-1 positive gene were performed using conjugation, PCR-based replicon typing, and whole-genome sequencing. A total of 22 isolates with acquired colistin resistance were identified giving an overall prevalence of 12.2% (22/181): 17 Escherichia coli (predominantly ST224 [n = 4, 23.5%]) and 5 Klebsiella pneumoniae (ST17 [n = 2, 40%], ST646 [n = 2, 40%], and ST944 [n = 1, 20%]). mcr-1 gene was exclusively found in 11 E. coli (prevalence of 6.1% [11/181]) and was associated with IncFV (n = 7) and IncFIIK (n = 4) plasmids. All the isolates had a commensal origin (n = 11). One isolate harbored virulence profile, a high colistin resistance (minimum inhibitory concentration = 96 mg/L), with some new mutations in the chromosomic colistin-resistant genes and different pathogenicity islands typically identified in uropathogenic E. coli. Conclusions: This study reports the diffusion of mcr-1 producing Enterobacterales from chicken meat in Western Algeria. This represents a worrisome situation needing continuous monitoring.