Repetitive Extragenic Palindromic PCR Research Articles

Francisella salimarina infection in farmed cobia (Rachycentron canadum) and dusky grouper (Epinephelus marginatus) in Brazil: A case report.

This study aimed to identify and characterize isolates of Francisella salimarina associated with an outbreak on a marine fish farm in Brazil and to analyse their genetic variability and antimicrobial susceptibility. In 2021, diseased cobias (Rachycentron canadum, n = 10) and dusky groupers (Epinephelus marginatus, n = 10) were sampled and subjected to bacteriological and pathological examinations. The isolates obtained were morphologically and biochemically characterized and identified by matrix-assisted laser desorption/ionization-time of flight (MALDI-ToF) and 16S rRNA gene sequencing. The genetic diversity of these isolates was analysed using repetitive extragenic palindromic-polymerase chain reaction (REP-PCR). Antimicrobial susceptibility was assessed using the disk diffusion technique. Macroscopically, the fish presented skin ulcerations, ocular lesions, hepatomegaly and splenomegaly. A pleomorphic, gram-negative, catalase- and oxidase-positive bacterium was isolated from seven cobias and two groupers. The 16S rRNA gene sequences showed >99% coverage and identity with other deposited sequences of F. salimarina. The results of the biochemical analysis corresponded to these bacterial species. Histologically, granulomas were observed in the spleen, liver and heart of the cobias (n = 6), and necrotizing and fibrinous dermatitis and myositis were identified in some groupers (n = 2). The isolates exhibited the same banding pattern when REP-PCR was performed, indicating that they were clonally related. Finally, the antibiogram test, no inhibition halo was observed for amoxicillin and trimethoprim-sulfamethoxazole. To our knowledge, this is the first report of F. salimarina infection in cobias and dusky groupers.

A comparative study of genotyping and antimicrobial resistance between carbapenem-resistant Klebsiella pneumoniae and Acinetobacter baumannii isolates at a tertiary pediatric hospital in China.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) clinical isolations have rapidly increased in pediatric patients. To investigate a possible health care-associated infections of CRKP in a tertiary pediatric hospital, the circulating clones and carbapenem-resistant pattern between CRKP and carbapenem-resistant Acinetobacter baumannii (CRAB) isolates were compared to classify their epidemiological characteristics. The results will help to identify the epidemic pattern of the CRKP transmission in the hospital. Ninety-six CRKP and forty-eight CRAB isolates were collected in Kunming Children's Hospital from 2019 through 2022. These isolates were genotyped using repetitive extragenic palindromic-PCR (REP-PCR). Carbapenemase phenotypic and genetic characterization were investigated using a disk diffusion test and singleplex PCR, respectively. In addition, these characteristics of the two pathogens were compared. The rates of CRKP and CRAB ranged from 15.8% to 37.0% at the hospital. Forty-nine and sixteen REP genotypes were identified among the 96 and 48 CRKP and CRAB isolates tested, respectively. The CRKP isolates showed more genetic diversity than the CRAB isolates. Of the 96 CRKP isolates, 69 (72%) produced Class B carbapenemases. However, all 48 CRAB isolates produced Class D carbapenemase or extended-spectrum β-lactamases (ESBL) combined with the downregulation of membrane pore proteins. Furthermore, the carbapenemase genes bla KPC, bla IMP, and bla NDM were detected in CRKP isolates. However, CRAB isolates were all positive for the bla VIM, bla OXA-23, and bla OXA-51 genes. These CRKP isolates exhibited different biological and genetic characteristics with dynamic changes, suggesting widespread communities. Continuous epidemiological surveillance and multicenter research should be carried out to strengthen the prevention and control of infections.

Plant growth promoting activities of Pseudomonas sp. and Enterobacter sp. isolated from the rhizosphere of Vachellia gummifera in Morocco.

The Moroccan endemic Vachellia gummifera grows wild under extreme desert conditions. This plant could be used as an alternative fodder for goats, and camels, in order to protect the Argan forests against overgrazing in Central and Southwestern Moroccan semiarid areas. With the aim to improve the V. gummifera population's density in semiarid areas, we proposed its inoculation with performing plant growth-promoting bacteria. Hence, 500 bacteria were isolated from the plant rhizosphere. From these, 291 isolates were retained for plant growth-promoting (PGP) activities assessment. A total of 44 isolates showed the best phosphates solubilization potential, as well as siderophore and auxin production. The combination of REP-PCR (repetitive extragenic palindromic-polymerase chain reaction) fingerprinting, PGP activities, and phenotypic properties, allowed the selection of three strains for the inoculation experiments. The three selected strains' 16S rRNA sequencing showed that they are members of the Enterobacter and Pseudomonas genera. The inoculation with three strains had diverse effects on V. gummifera growth parameters. All single and combined inoculations improved the plant shoot weight by more than 200%, and the root length by up to 139%, while some combinations further improved protein and chlorophyll content, thereby improving the plant's forage value. The three selected strains constitute an effective inoculum for use in the arid and semiarid zones of southern Morocco.

Edwardsiella tarda in Tambaqui (Colossoma macropomum): A Pathogenicity, Antimicrobial Susceptibility, and Genetic Analysis of Brazilian Isolates.

Edwardsiella tarda is a crucial pathogenic bacterium in tropical aquaculture. This bacterium was recently isolated from tambaqui (Colossoma macropomum), a commercially important fish species in Brazil. This study assessed the antimicrobial susceptibility, pathogenicity, and genetic diversity of the tambaqui-derived E. tarda isolates. Fourteen bacterial isolates isolated from tambaqui were identified as E. tarda by using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry and dnaJ gene sequencing. Antimicrobial susceptibility tests were conducted against seven drugs using the disc diffusion assay. The pathogenicity test conducted by intraperitoneal injection of 2.4 × 107 colony-forming units (CFU) fish-1 of E. tarda (ED38-17) into tambaqui juveniles eventually revealed that neither clinical signs nor death were present. However, splenomegaly and whitish areas in the spleen and kidneys were observed. The histological investigation also revealed granulomatous splenitis, nephritis, and hepatitis occurring internally. Repetitive extragenic palindromic-PCR fingerprinting separated the 14 isolates into three genetic groups. The antibiogram revealed that all E. tarda isolates were wild-type (WT) to florfenicol (FLO), norfloxacin (NOR), neomycin (NEO), erythromycin (ERY), and oxytetracycline (OXY); however, some were non-wild-type to sulfamethoxazole/trimethoprim (7.1%) and amoxicillin (21.4%). Therefore, through experimental infection, E. tarda ED38-17 could induce pathogenic effects in C. macropomum. Additionally, three distinct genetic types were found, and the E. tarda isolates were WT to FLO, NOR, NEO, ERY, and OXY. These findings raise awareness of a bacteria causing unseen lesions, a pathogen that will potentially impact tambaqui aquaculture in the future.

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.

Outbreak of Burkholderia multivorans among patients at two acute-care hospitals in California, August 2021–July 2022

Background:Burkholderia multivorans are gram-negative bacteria typically found in water and soil. B. multivorans outbreaks among patients without cystic fibrosis have been associated with exposure to contaminated medical devices or nonsterile aqueous products. Acquisition can also occur from exposure to environmental reservoirs like sinks or other hospital water sources. We describe an outbreak of B. multivorans among hospitalized patients without cystic fibrosis at 2 hospitals within the same healthcare system in California (hospitals A and B) between August 2021 and July 2022. Methods: We defined confirmed case patients as patients without cystic fibrosis hospitalized at hospital A or hospital B between January 2020 to July 2022 with B. multivorans isolated from any body site matching the outbreak strain. We reviewed medical records to describe case patients and to identify common exposures. We evaluated infection control practices and interviewed staff to detect exposures to nonsterile water. Select samples from water, ice, drains, and sink splash zone surfaces were collected and cultured for B. multivorans in March 2022 and July 2022 from both hospitals. Common aqueous products used among case patients were tested for B. multivorans. Genetic relatedness between clinical and environmental samples was determined using random amplified polymorphic DNA (RAPD) and repetitive extragenic palindromic polymerase chain reaction (Rep-PCR). Results: We identified 23 confirmed case patients; 20 (87%) of these were identified at an intensive care unit (ICU) in hospital A. B. multivorans was isolated from respiratory sources in 18 cases (78%). We observed medication preparation items, gloves, and patient care items stored within sink splash zones in ICU medication preparation rooms and patient rooms. Nonsterile water and ice were used for bed baths, swallow evaluations, and ice packs. B. multivorans was cultured from ice and water dispensed from an 11-year-old ice machine in the ICU at hospital A in March 2022 but no other water sources. Additional testing in July 2022 yielded B. multivorans from ice and a drain pan from a new ice machine in the same ICU location at hospital A. All products were negative. Clinical and environmental isolates were the same strain by RAPD and Rep-PCR. Conclusions: The use of nonsterile water and ice from a contaminated ice machine contributed to this outbreak. Water-related fixtures can serve as reservoirs for Burkholderia, posing infection risk to hospitalized and immunocompromised patients. During outbreaks of water-related organisms, such as B. multivorans , nonsterile water and ice use should be investigated as potential sources of transmission and other options should be considered, especially for critically ill patients.Disclosures: None

Hospital-acquired infections due to carbapenem-resistant Providencia stuartii.

During the course of a retrospective survey on healthcare associated infections (HAIs) due to carbapenem-resistant organisms, an unusual prevalence of HAIs due to carbapenem-resistant Providencia stuartii (CRPS) was found. Hence this study aimed to conduct the occurrence of P. stuartii associated HAIs with special reference to the drug resistance profiling of these isolates. Of the eight total HAI cases (7.5% of total HAIs and 33.3% of HAIs due to Enterobacterales) of CRPS infections included in this study, three were reported from ventilator-associated pneumonia (VAP), three were surgical site infections (SSIs), one was a catheter-associated urinary tract infection (CAUTI) and one was a bloodstream infection. All the eight CRPS isolates were tested for extended-spectrum β-lactamases production, AmpC hyperproduction as well as carbapenem resistance. Typing of the isolates was performed by repetitive extragenic palindromic polymerase chain reaction (REP-PCR). All the eight isolates of CRPS were found to be AmpC hyperproducers, carbapenemase producers, and harboured chromosomally located blaNDM in seven isolates and blaIMP genes in one. All the cases with CRPS infections had prior history of colistin therapy along with prolonged hospital stay (>20 days). The cases were located in five different wards/intensive care unit (ICU) within the hospital in one year. However, strain typing by REP-PCR revealed 100 per cent similarity and clonal relatedness in all the seven isolates carrying blaNDM genes. Interestingly, routine hospital surveillance revealed a high carriage of P. stuartii in the axilla of patients admitted to the ICU. The study findings suggest CRPS as an important cause of HAIs. This organism often goes unnoticed due to the burden of carbapenem resistance in other Enterobacterales and non-fermenters.

Distribution and Characteristics of Bacteria Isolated from Cystic Fibrosis Patients with Pulmonary Exacerbation.

Cystic fibrosis (CF) is an inherited recessive disorder characterized by recurrent and persistent pulmonary infections, resulting in lung function deterioration and early mortality. A cross-sectional study was conducted on the bacterial profile and antibiotic resistance pattern of 103 respiratory specimens from CF patients with signs of pulmonary exacerbation. Antibiotic susceptibility testing and biofilm formation of Staphylococcus aureus and Pseudomonas aeruginosa isolates were performed by the Kirby-Bauer disc diffusion method and microtiter plate assay, respectively. Molecular typing of S. aureus and P. aeruginosa isolates was carried out by spa typing and repetitive extragenic palindromic element PCR. In a total of 129 isolates, the most prevalent organisms were S. aureus (55.3%) and P. aeruginosa (41.7%). Other less prevalent bacterial isolates include coagulase-negative staphylococci, Escherichia coli, klebsiella spp., Enterobacter spp., and Achromobacter xylosoxidans. The highest rate of resistance for S. aureus was observed to azithromycin and erythromycin (80%), ciprofloxacin (52.3%), clindamycin (44.6%) and tetracycline (43%). Twenty percent of S. aureus isolates were methicillin-resistant S. aureus (MRSA) and 47.6% were MDR S. aureus. For P. aeruginosa isolates the highest resistance was to cefepime (38.3%) and levofloxacin (33.3%) and 20% showed MDR phenotype. Our study demonstrated a significant decline in the prevalence of P. aeruginosa infections in comparison to previous studies. We found S. aureus to be more prevalent in younger patients, whereas mucoid P. aeruginosa showed a shift in prevalence toward older ages. Molecular typing methods showed great diversity between isolates.

Reservoir of Carbapenem-Resistant Acinetobacter baumannii in the Hospital Environment and Colonization Pressure: A Surveillance-Based Study in Indian Intensive Care Unit.

The endemicity of Acinetobacter baumannii in intensive care units (ICUs) is a serious concern. We studied the reservoirs of A. baumannii in the ICU and their effects on colonization pressure and transmission. A prospective surveillance (6 months) was conducted. Screening culture (rectal and axillary) swabs were collected within 48 hours admission and in 120 hours. Surveillance cultures from patients' surroundings, health care workers (HCWs), and hospital sewage were collected. A. baumannii was identified by phenotypic and genotypic methods. Carbapenem resistance and insertion sequence element were detected. Typing was done by repetitive extragenic palindromic-polymerase chain reaction and multilocus sequence typing. Colonization pressure was calculated and compared with environment colonizers. Of the 87 patients, 21.83% (19) were colonized with A. baumannii, 73.68% (14/19) were imported, and 26.31% (5/19) acquired carriers. Axilla was the commonest site. From the environment (15), bed rails 33.33% (5/15) and suction tubes 26.66% (4/15) were the common sites. HCWs showed 7.5% (3/40) carriage. Carbapenem resistance with blaOXA-51, blaOXA-23, and ISAba1 were 91.89% (34/37). Strong correlation between colonization pressures and environmental colonizers was seen (r2 = 0.719, p = 0.032). Carbapenem and polymyxin B were (p ≤ 0.05) significant exposures. Sequence type 623 was the predominant cluster with isolates from carriers, HCWs, and environment. Colonization pressure of carbapenem-resistant A. baumannii depends on their presence in the hospital. Hands of HCWs were an important vehicle for transmission. Infection control measure should consider reducing the environmental reservoir.

First Report of Bacterial Blight on arugula (Eruca vesicaria subsp. sativa) caused by Pseudomonas cannabina pv. alisalensis in New York.

Bacterial blight of arugula (Eruca vesicaria subsp. sativa cv. Standard) was observed in a commercial crop being grown in a high tunnel under overhead irrigation in Argyle, NY in January 2021. Approximately 80-100% of the plants were affected. Symptoms started as small, angular, water-soaked lesions visible on both sides of the leaves, then expanded, coalesced and later dried and turned tan. Fluorescent pseudomonads from five different plants were isolated on King's medium B agar amended with boric acid, chloramphenicol, and cycloheximide (Schaad et al. 2001) from surface disinfested symptomatic leaf tissues macerated in phosphate buffer (10mM, pH 7.0). Five representative isolates from each of the five plants produced levan and were negative for arginine dihydrolase and oxidase. They did not rot potatoes but were able to induce a hypersensitive reaction on tobacco (Nicotiana tabacum L. cv Glurk) within 24 h, thus demonstrating that the isolates belonged to LOPAT group 1 (Lelliott et al. 1966). DNA fragment banding patterns of these isolates generated by repetitive extragenic palindromic sequence PCR (repPCR) with BOXA1R primers were compared to the pathotype strains of Pseudomonas cannabina pv. alisalensis and Pseudomonas syringae pv. maculicola, known fluorescent pathogens of the Brassicaceae. The repPCR banding pattern of all isolates matched the pattern of Pseudomonas cannabina pv. alisalensis. Bacterial inoculum for pathogenicity experiments was prepared from 48 h KBBC agar cultures suspended in phosphate buffer (10mM, pH 7.0) and adjusted to 0.6 optical density at 600nm yielding approximately 108 CFU/ml. Five-week-old arugula plants were sprayed until run-off with one of the three isolates from arugula, a negative control (sterile buffer), or a positive control (P. cannabina pv. alisalensis) that is pathogenic to crucifers and also reported to cause disease on arugula in California (Bull et al. 2004). Experiments consisted of three replications of each treatment and two independent experiments were conducted. Small water-soaked spots resembling the original symptoms developed on all plants inoculated with the three representative isolates and P. cannabina pv. alisalensis. Moreover, reisolates from the symptomatic tissues were fluorescent on KBBC and had identical repPCR banding pattern as inoculated strains, demonstrating Koch's postulates. To our knowledge, this is the first report of bacterial blight on arugula caused by Pseudomonas cannabina pv. alisalensis in the Northeastern US. It was previously reported in California, Nevada, and Minnesota (Bull and du Toit 2009; Bull et al. 2004). This report may have significance for all brassica leafy green growers in the Northeast as P. cannabina pv. alisalensis has a broad host range including members of the Brassicaceae and oats which are commonly used as cover crops in mixed vegetable production.

Genotypic and phenotypic comparison of clinical and environmental Acinetobacter baumannii strains

The genotypic and phenotypic characteristics and antibiotic resistance (antibiogram) profiles of clinical (n = 13) and environmental (n = 7) Acinetobacter baumannii isolates were compared. Based on the Repetitive Extragenic Palindromic Sequence-based PCR (REP-PCR) analysis, the clinical and environmental A. baumannii isolates shared low genetic relatedness (∼60%). Multilocus sequence typing (MLST, Oxford scheme) indicated that the clinical A. baumannii were assigned to three sequence types (ST231, ST945 and ST848), while the environmental A. baumannii (excluding AB 14) were categorised into the novel ST2520. The majority of the clinical (excluding AB 5, CAB 11, CAC 37) and environmental (excluding AB 14 and AB 16) A. baumannii strains were then capable of phase variation with both the translucent (71.4%; 15/21) and opaque (95.2%; 20/21) colony phenotypes detected. The clinical isolates however, exhibited significantly (p < 0.05) higher biofilm formation capabilities (OD570: 2.094 ± 0.497). Moreover, the clinical isolates exhibited significantly (p < 0.05) higher resistance to first line antibiotics, with 92.3% (12/13) characterised as extensively drug resistant (XDR), whereas environmental A. baumannii exhibited increased antibiotic susceptibility with only 57.1% (4/7) characterised as multidrug resistant (MDR). The environmental isolate AB 14 was however, characterised as XDR. In addition, only five clinical A. baumannii isolates exhibited colistin resistance (38.5%; 5/13). The current study highlighted the differences in the genotypic, phenotypic, and antibiotic resistance profiles of clinical and environmental A. baumannii. Moreover, the environmental strains were assigned to the novel ST2520, which substantiates the existence of this opportunistic pathogen in extra-hospital reservoirs.

Authentication of putative competitive bacterial endophytes of rice by re-isolation and DNA fingerprintingassay.

The plant-growth-promoting putative competitive endophytes offer significant benefits to sustainable agriculture. The unworthy opportunistic and passenger endophytes are inevitable during the isolation of putative competitive endophytes. This study aimed to discriminate the putative competitive endophytes undoubtedly from the opportunistic and passenger endophytes. The newly isolated endophytes from field-grown rice were inoculated to 5-days old rice seedlings under gnotobiotic conditions. Re-isolation of the inoculated strains from the root surface, inner tissues of the whole plant, root and shoot was performed after 5-days. All the re-isolated colonies were compared with native isolates for homology by BOX-A1R-based repetitive extragenic palindromic-PCR (BOX-PCR) and enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) DNA fingerprints. The results revealed that the putative competitive endophytes (RE25 and RE10) showed positive for re-isolation and BOX and ERIC fingerprints for the whole plant, root and shoot. The opportunistic (RE27 and RE8) and passenger endophytes (RE44 and RE18) failed in re-isolation either from root or shoot. The epiphytes (ZSB15 and Az204) showed negative for endophytic re-isolation and positive for surface colonization. This modified procedure can discriminate the putative competitive endophytes from others. Eliminating the opportunistic and passenger endophytes and epiphytes early by this method would help develop endophytic inoculants to enhance rice productivity.

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.

Further culture-independent characterization of the lactic microbiota of Serro artisanal cheese.

This study aimed to provide a further characterization of the lactic microbiota present in Minas artisanal cheese (MAC) from the Serro region by using culture-independent methods, as a complementary analysis of a previous study. The total DNA extracted from MAC samples (n = 55) was subjected to repetitive extragenic palindromic-PCR (rep-PCR) and PCR-denaturing gradient gel electrophoresis (PCR-DGGE). Rep-PCR analysis showed that core microbiota of Serro MAC was closely related, independent of the production town, farm size, or time of production. The sequencing of PCR-DGGE bands identified the prevalence of Lactococcus lactis in all samples, and Streptococcus salivarius was also identified. Thus, we conclude that when more accurate methods are unavailable, rep-PCR can be used as a culture-independent method to demonstrate if the microbiota is closely related or not among the samples. PCR-DGGE results also matched to the main findings of high-throughput sequencing, previously presented, confirming its confidence to detect the main microbial groups present in the raw milk cheeses.

Genotypic and phenotypic characterization of Escherichia coli isolated from indigenous individuals in Malaysia.

Objective(s):The occurrence of asymptomatic verocytotoxin (VT)-producing Escherichia coli (VTEC) infections among humans in recent years is posing a high risk to public health. Thus, the role of asymptomatic human carriers as a source of dissemination should not be underestimated. This study aimed to elucidate the phenotypic and genotypic characteristics of E. coli in the stool samples collected from indigenous individuals in Malaysia. Materials and Methods: E. coli strains (n=108) were isolated from stool samples obtained from 41 indigenous individuals. All strains were subjected to Repetitive Extragenic Palindromic-Polymerase Chain Reaction (REP-PCR) typing and confirmation of VTEC variants. Non-duplicate strains were selected based on REP-PCR profiles and further subjected to antimicrobial susceptibility test (AST). The genotypic and phenotypic characteristics of the strains were then correlated with the demographic data of the subjects. Results:A total of 66 REP-PCR profiles grouped in 53 clusters (F=85%) were obtained. Four genetically distinct strains were confirmed as VTEC (eaeA-positive). The predominant resistance was against ampicillin (34.2%), followed by trimethoprim-sulfamethoxazole (32.9%), ampicillin-sulbactam (5.5%), and ciprofloxacin (1.4%). All isolates were sensitive to amoxicillin-clavulanate, cefuroxime, ceftriaxone, imipenem, and meropenem. Conclusion:Genetically diverse E. coli and VTEC strains were found to colonize the intestines of the indigenous populations. This study is important for the prospective surveillance of E. coli among the indigenous individuals in Malaysia, especially in asymptomatic VTEC infection and antimicrobial resistance phenomenon.

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.

Phase variation in the glycosyltransferase genes of Pasteurella multocida associated with outbreaks of fowl cholera on free-range layer farms.

Fowl cholera caused by Pasteurella multocida has re-emerged in Australian poultry production since the increasing adoption of free-range production systems. Currently, autogenous killed whole-cell vaccines prepared from the isolates previously obtained from each farm are the main preventative measures used. In this study, we use whole-genome sequencing and phylogenomic analysis to investigate outbreak dynamics, as well as monitoring and comparing the variations in the lipopolysaccharide (LPS) outer core biosynthesis loci of the outbreak and vaccine strains. In total, 73 isolates from two different free-range layer farms were included. Our genomic analysis revealed that all investigated isolates within the two farms (layer A and layer B) carried LPS type L3, albeit with a high degree of genetic diversity between them. Additionally, the isolates belonged to five different sequence types (STs), with isolates belonging to ST9 and ST20 being the most prevalent. The isolates carried ST-specific mutations within their LPS type L3 outer core biosynthesis loci, including frameshift mutations in the outer core heptosyltransferase gene (htpE) (ST7 and ST274) or galactosyltransferase gene (gatG) (ST20). The ST9 isolates could be separated into three groups based on their LPS outer core biosynthesis loci sequences, with evidence for potential phase variation mechanisms identified. The potential phase variation mechanisms included a tandem repeat insertion in natC and a single base deletion in a homopolymer region of gatG. Importantly, our results demonstrated that two of the three ST9 groups shared identical rep-PCR (repetitive extragenic palindromic PCR) patterns, while carrying differences in their LPS outer core biosynthesis loci region. In addition, we found that ST9 isolates either with or without the natC tandem repeat insertion were both associated with a single outbreak, which would indicate the importance of screening more than one isolate within an outbreak. Our results strongly suggest the need for a metagenomics culture-independent approach, as well as a genetic typing scheme for LPS, to ensure an appropriate vaccine strain with a matching predicted LPS structure is used.

Heterogeneity of Yersinia ruckeri Isolated from Rainbow Trout (Oncorhynchus mykiss) Infected with Enteric Red Mouth Disease

Regarding the increasing prevalence of antimicrobial resistance as a global menace, typing procedures are of great importance in epidemiological surveys. In the current study, interspecies differences of Yersinia ruckeri (Y. ruckeri) isolates were detected by antimicrobial susceptibility profiling and molecular traits to determine the relationship between isolates. The current study was conducted on 27 Y. ruckeri isolates collected from 40 infected rainbow trout (Oncorhynchus mykiss) with clinical enteric red mouth disease in Mashhad, Iran. Interspecies differences of all isolates were detected by antimicrobial susceptibility profiling using disk diffusion method, Repetitive Extragenic Palindromic PCR (REP-PCR), Enterobacterial Repetitive Intergenic Consensus PCR (ERIC-PCR), and Random Amplification of Polymorphic DNA PCR (RAPD-PCR) to determine the relationship between isolates. Simpson’s diversity index was calculated for each typing technique. The most phenotype resistant was against ampicillin, chloramphenicol, and lincomycin. Furthermore, enrofloxacin and ciprofloxacin showed the highest activity (100%) against isolates. According to the results, the highest diversity index was observed in the REP-PCR method (D = 0.91). In conclusion, REP-PCR can be a powerful technique for epidemiological studies.

Associative bacterial diversity of pangolão, a stressresilient tropical grass

Some forage species, such as pangolão grass (Digitaria eriantha Steud. cv. Survenola), are resilient in tropical semi arid regions. A possible reason for this is the presence of endophytic and rhizospheric microorganisms. Thus, this study evaluated the diversity of associative bacteria in pangolão grass. Bacteria associated with the roots, culm, leaves, and rhizospheric soil were isolated and characterized in three municipalities of Pernambuco, Brazil. An initial phenotypic characterization was followed by a genotypic assessment by based repetitive extragenic palindromic-polymerase chain reaction (BOX-PCR) and partial sequencing of the 16S rRNA gene. We obtained 325 phenotypically-characterized isolates grouped into 243 strains with 100% similarity by BOX-PCR. The most diverse sampling environment was Araripina, and all factors affected bacterial diversity. There were 135 groups with 90% similarity, that were represented by a single strain each for sequencing. Among the sequenced strains, 118 showed 96.84–99.9% similarity with previously described strains, whereas 17 could not be identified. The following 18 genera were identified from three phyla, five classes, seven orders, and 13 families: Achromobacter, Agrobacterium, Bacillus, Burkholderia, Curtobacterium, Enterobacter, Herbaspirillum, Kosakonia, Ochrobactrum, Paenibacillus, Pantoea, Priestia, Pseudomonas, Rhizobium, Serratia, Shinella, Stenotrophomonas, and Variovorax. The diversity of endophytic and rhizospheric bacteria may contribute to the resilience of pangolão, as various strains of these genera have been described as plant growth promoters. This is the first evaluation of pangolão bacterial diversity under tropical semi arid conditions. Since several of the genera include strains known to promote plant growth, we propose further research to evaluate this on crops.

High Prevalence of blaCTX-M in Fecal Commensal Escherichia coli from Healthy Children.

BackgroundAntibiotic-resistant Escherichia coli can colonize the intestinal tract of healthy children, causing concern when antibiotic resistance is related to the presence of transferable mechanisms, such as extended-spectrum β-lactamases (ESBLs).Materials and MethodsFecal samples from 41 healthy children from two villages of rural Peru were cultured on ceftriaxone-disks. ESBL production was confirmed with double disk synergy. In all ESBL-produced isolates, antibiotic susceptibility to 12 antibacterial agents was established by disk diffusion, while clonal relationships were determined by repetitive extragenic palindromic-polymerase chain reaction (REP-PCR). Presence of ST131 was determined using PCR.ResultsCeftriaxone-resistant microorganisms were recovered from 39 samples belonging to 22 out of 41 children (53.7%). Of these, 80 ceftriaxone-resistant and two ceftriaxone-intermediate E. coli from inside ceftriaxone-halos were confirmed as ESBL-producers. All isolates were multidrug-resistant. In 79/80 (98.8%) ceftriaxone-resistant isolates, the presence of blaCTX-M was detected alone (58 isolates, or together with other β-lactamase (blaTEM, 17 isolates; blaOXA-1-like, 3 isolates; blaTEM + blaOXA-1-like, 1 isolate), while in one isolate no such ESBL was identified. The two ceftriaxone-intermediate isolates recovered from the same sample, carried a blaTEM and blaSHV respectively. Thirty-four different clones were identified, with 4 clones being recovered from different samples from the same child. Twelve clones were disseminated among different children, including 5 clones disseminated between both villages. Two clones, accounting for 3 isolates and both recovered from the same children, belonged to E. coli ST131.ConclusionThis study demonstrates high prevalence of ESBL-carriers among healthy children living in a rural area of Peru, stressing the need for continuous surveillance and search for public health control measures.