Mucoid Colonies Research Articles

Prevalence and Antibiotic Resistance of Klebsiella pneumoniae in Diabetic Foot Ulcer.

Introduction A serious global threat of antimicrobial resistance has emerged due tothe improper use of antibiotics, including polypharmacy and inappropriate prescribing. This misuse has led bacteria to develop immunity against these drugs.Klebsiella pneumoniae, a concerning gram-negative bacterium, has become resistant, especially among immunocompromised diabetic patients for multiple antibiotics. To fight effectively this growing crisis and regain control of these infections, it is crucial to comprehend the resistance mechanisms utilized by the bacteria anddevelop a new therapeutic strategy to prevent antibiotic resistance. Materials and methods A five-month study from January 2023 to May 2023 was conducted at the tertiary healthcare facility of Saveetha Medical Collegeby collecting 122 clinical specimens from patients with diabetic foot ulcers (DFUs) and ulcer-related infections. The microbiological testing methods followed by the identification of bacteria using matrix-assisted laser desorption/ionizationtime-of-flight mass spectrometry (MALDI-TOF) and antimicrobial susceptibility testing (AST) by the VITEK 2 Compact system were performed. Results A stout, rod-shaped, gram-negative bacilli was observed in gram staining, and growth of mucoid γ-hemolytic colonies and lactose-fermenting mucoid colonies were seen in blood and MacConkey agar plates. MALDI-TOF analysis confirmed the presence ofKlebsiella pneumoniaealong with other bacteria such as Klebsiella oxytoca, Staphylococcus spp., Proteus spp., and​​Escherichia ​coli. VITEKshowed high resistance rates to commonly used antibiotics, including carbapenems. Notably, isolates showed sensitivity and intermediate to tigecycline and colistin. Resistance patterns varied across specimen types, emphasizing the importance of considering clinical sources when interpreting data. Conclusion Hence, this study underscores the urgent need for novel antimicrobial agents and effective infection control measures to combat multidrug-resistant Klebsiella pneumoniae infections. Understanding resistance mechanisms is essential for the incorporation of treatment strategies and preserving antibiotic efficacy.

Morphological and biochemical identification of Ralstonia solanacearum strains in ginger (Zingiber officinale Roscoe) wilt disease

The Ralstonia solanacearum Species Complex (RSSC) is a significant plant pathogen affecting various agricultural commodities, including ginger. Understanding the distribution and characteristics of RSSC in ginger plants is crucial for effective disease management. This study aims to determine the distribution of RSSC in ginger plants and identify the pathogen through morphological and biochemical characterization. The research was conducted in Banyubiru and Getasan districts, Semarang Regency, Indonesia. The study involved purposive sampling, isolation, and purification of isolates, followed by morphological characterization through observation and biochemical characterization using Gram staining with KOH, oxidase test, arginine activity test, hypersensitivity test, bacterial density calculation, Koch's postulates, and biovar characterization. Three isolates from Banyubiru displayed typical morphological characteristics of RSSC, including round, mucoid colonies with red centers and white edges on TZC medium, and rod-shaped bacterial cells. Biochemical characterization identified these isolates as RSSC strains biovar 3 and 4, capable of causing bacterial wilt in ginger plants. The study confirmed the presence of bacterial wilt in ginger in Banyubiru district. The findings reveal the spread of bacterial wilt caused by RSSC in ginger plants in Banyubiru, Semarang Regency. There is a need for measures to control the potential spread of RSSC in the surrounding host plant areas in Tlumpak Village, Banyubiru district.

Genomic Comparative of Pseudomonas aeruginosa Small Colony Variant, Mucoid and Non-mucoid Phenotypes Obtained from a Patient with Cystic Fibrosis During Respiratory Exacerbations.

Pseudomonas aeruginosa, the most prevalent opportunistic pathogen in chronic obstructive pulmonary disease, associated with high morbidity and mortality in patients with cystic fibrosis (CF), is practically impossible to be eradicated from the airways in chronicity. Its extraordinary genomic plasticity is possibly associated with high antimicrobial resistance, virulence factors, and its phenotypic diversity. The occurrence of P. aeruginosa isolates promoting airway infection, showing mucoid, non-mucoid, and small colony variant (SCV) phenotypes, was observed simultaneously, in the present study, in sputum cultures obtained from a male CF young patient with chronic pulmonary infection for over a decade. The isolates belonged to a new ST (2744) were obtained in two moments of exacerbation of the respiratory disease, in which he was hospitalized. Genetic background and phenotypic analysis indicated that the isolates exhibited multi- and pan-antimicrobial resistant profiles, as well as non-susceptible to polymyxin and predominantly hypermutable (HPM) phenotypes. Whole genome sequencing showed variations in genome sizes, coding sequences and their determinants of resistance and virulence. The annotated genomes were compared for antimicrobial resistance, hypermutability, and SCV characteristics. We highlight the lack of reported genetic determinants of SCV emergence and HPM phenotypes, which can be explained in part due to the very short time between collections of isolates. To the best of our knowledge, this is the first report of genome sequencing of P. aeruginosa SCV from a CF patient in Brazil.

First Report of Xanthomonas perforans Causing Bacterial Spot of Pepper (Capsicum annuum) in North Carolina.

North Carolina (NC) is the fifth largest producer of bell pepper (Capsicum annuum) in the US with an estimated 2,400 acres in production (NASS-USDA, 2022). A survey of bacterial diseases of peppers was initiated in 2020 after numerous bacterial spot outbreaks were reported in NC. Bacterial spot is caused by a complex of four Xanthomonads: X. euvesicatoria, X. vesicatoria, X. perforans, and X. hortorum pv. gardneri (Larrahondo-Rodríguez et al., 2022). If not preemptively managed, bacterial spot can cause up to 40% yield loss (Kousik and Ritchie, 1998). During the 2020 and 2021 growing seasons, 103 yellow mucoid colonies were isolated from plants representing 51 pepper cultivars symptomatic of bacterial spot, i.e., water-soaked leaf lesions that become necrotic spots on leaves and fruits across 22 commercial fields in NC following published methods (Klein-Gordon et al., 2021). All colonies were characterized to species using the qPCR species-specific primers and probes described by Strayer et al. 2016. Of the 103 colonies, 12 isolates tested positive for X. perforans. To confirm qPCR results, a Multi-Locus Sequence Analysis (MLSA) was run using fusA, gapA, gltA, gyrB, and lacF following previously described methods (Almeida et al., 2010) on three representative isolates: AHX61, collected in September 2020 from a field with 20% disease severity in Wake County on cv. Canary Bell; AHX261, collected in July 2021 from a field with 50% disease severity in Sampson County on Jalapeño; and AHX426, collected in August 2021 from a field with 50% disease severity in Dublin County on Jalapeño. All gene sequences were deposited to NCBI (GenBank Accessions: OQ799538-OQ799552) and compared to those from X. euvesicatoria, X. hortorum pv. gardneri, X. perforans, and X. vesicatoria type strains (Almeida et al., 2010). The MLSA showed AHX61, AHX261, and AHX426 cluster with X. perforans ICMP16690T, sharing 99-100% nucleotide similarity. Koch's postulates were performed with the three strains, Xp1484T [ X. perforans type strain, (Wilson 1987)], and water as a negative control. Three 10-week-old bell pepper plants (cv. Early Cal Wonder) were dip-inoculated in 600 mL of a bacterial suspension at an OD600 of 0.3 (~5x108 CFU/mL) and 0.04% Silwet L-77 per strain or water. All 18 plants were individually incubated in a plastic bag for 48 h post-inoculation at 28°C, 80% relative humidity, and 14 h:10 h light-dark cycle in a growth chamber, after which plastic bags were removed. Water-soaking and necrotic spots characteristic of bacterial spot were first observed at six days post-inoculation (dpi). At 14 dpi, symptomatic leaves were removed from treated plants to attempt pathogen re-isolation. Yellow mucoid colonies similar in morphology to those originally inoculated were recovered from all plants and confirmed to be X. perforans through sequencing; no isolates were recovered from water-treated plants. To our knowledge, this is the first time X. perforans is isolated in commercial bell pepper and specialty pepper fields in the state. This is an indication that the Xanthomonas population on peppers in the state is more diverse than previously reported and that pathogen populations will require monitoring for possible species shifts for this crop in NC.

Bio-Prospecting Xylose-Utilizing, Exopolysaccharide (EPS)-Producing Bacteria and EPS Quantification through Submerged Fermentation using Xylose as the Major Carbon Source

Study’s Novelty/ Excerpt This study identified bacterial isolates from diverse environments capable of utilizing xylose for exo-polysaccharide (EPS) production, an area with limited prior research. Potential EPS producers were isolated and identified as Staphylococcus, Enterobacter, Kocuria, Klebsiella, Enterococcus, Serratia, and Burkholderia species using a salt-based medium with xylose as the primary carbon source. The highest EPS production was observed in Enterobacter cloacae D1, E. cloacae D2, Klebsiella oxytoca D2, and K. oxytoca G1, ranging from 0.04 g/L to 2.0 g/L, highlighting these isolates' potential for optimization and application in various industries. Full Abstract Many microorganisms are capable of producing Exo-polysaccharides (EPS) while utilizing simple sugars and hexoses. These EPS found applications in various fields, such as agricultural biotechnology, pharmaceuticals, textiles, and food industries. However, there is a lack of studies on EPS-elaborating bacteria that can utilize pentoses like xylose. Therefore, the utilization of alternative carbon sources for EPS production has become a focus of recent research. This study aimed to prospect bacteria that can utilize xylose for EPS production. Samples from agricultural soil, dump sites, saline soil, cement-contaminated soil, fresh cow milk, cow dung, and yogurt were serially diluted and cultured in a salt-based medium with xylose as the primary carbon source. Slimy and mucoid colonies were selected as potential EPS-producing isolates and identified morphologically and biochemically using the VITEK 2 Automated identification system. The quantification of EPS production by these isolates was conducted through submerged fermentation with xylose as the sole carbon source. The mean heterotrophic bacterial count of xylose-utilizing bacteria ranged from 2.1x106 CFU to 3.5x108 CFU per gram of the samples analyzed. The slimy and mucoid colonies were identified as members of the genera Staphylococcus, Enterobacter, Kocuria, Klebsiella, Enterococcus, Serratia, and Burkholderia. The quantities of EPS produced by the isolates ranged from 0.04 g/L to 2.0 g/L, with E. cloacae D1, E. cloacae D2, K. oxytoca D2, and K. oxytoca G1 elaborating the highest amount of EPS. Bacterial isolates capable of utilizing xylose for EPS production were obtained from various sources, showing potential for further optimization

Prevalence and antimicrobial mechanism of carbapenem-resistant Klebsiella pneumoniae and its molecular properties

BackgroundThe carbapenem-resistant Klebsiella pneumoniae poses a serious threat to public health because carbapenems are used as a final resort to treat K. pneumoniae-mediated infections in humans. MethodsSamples were collected from various clinical specimens including skin swabs, anal swabs, wound swabs, oral swabs, and sputum by the standard method and K. pneumoniae strains were isolated. Biofilm-forming characters were determined. The antibiotic resistance pattern was analyzed by the Kirby–Bauer disk diffusion method. Carbapenem resistance properties were tested using the imipenem and meropenem antibiotics. The carbapenemase genes (blaKPC and blaNDM) were determined. ResultsA total of 11 cephalosporin-resistant K. pneumoniae (CRKP) strains were isolated from the samples. The screened CRKP strains exhibited multi-drug resistance and non-susceptible to imipenem, ceftazidime, piperacillin, ceftriaxone, cefazolin, ampicillin, aztreonam, and cefotetan antibiotics. A total of 79.4 % K. pneumoniae isolates showed positive results on String-forming test. About 82.1 % of isolates showed mucoid colonies and 59 % of K. pneumoniae strains formed biofilm (p < 0.01). Out of 11 isolates, four strains exhibited Klebsiella pneumoniae carbapenemase type, three strains produced metallo-β-lactamases (MBL), and four strains exhibited as carbapenemase types. A total of 63.5 % Klebsiella pneumoniae carbapenemase-producing strains showed very low MIC value (<0.05 mg/L). ConclusionsDrug-resistance K. pneumoniae was isolated from clinical specimens that were screened. The continuous monitoring of drug-resistance genes are required to make policy decisions.

Antimicrobial susceptibility profile of Klebsiella pneumoniae isolated from some dairy products in Libya as a foodborne pathogen.

Klebsiella pneumoniae is one of the most common causes of clinical and asymptomatic mastitis in dairy cattle, as well as in milk and dairy products that affect milk quality. Mastitis caused by K. pneumoniae is even more serious due to its poor response to antibiotic therapy. The aim of this study was to detect and identify the presence of K. pneumoniae in milk and dairy products produced in Libya. A total of 234 samples were randomly collected from various locations in Libya. Samples were examined for the presence of K. pneumoniae using conventional cultural techniques, including cultivation in violet red bile agar plus 4-methylumbelliferyl-ß-D-glucuronide broth and CHROM agar, followed by polymerase chain reaction identification and partial sequencing of 16S rRNA. Of the 234 samples of milk and dairy products collected, 16 (6.8%) isolates revealed mucoid colonies on agar media that were phenotypically suggested to be K. pneumoniae. Identification of isolates was confirmed using molecular techniques (16S rRNA). Among the examined samples, K. pneumoniae was recovered from camel's milk, raw cow's milk, raw fermented milk, Maasora cheese, Ricotta cheese, soft cheese, full cream milk powder, milk powder infant formula, cereal baby food, and growing-up formula. Antibiotic susceptibility testing was performed on 12 of the 16 K. pneumoniae isolates, and the results showed that K. pneumoniae isolates were resistant to more than eight antibiotics; interestingly, two isolates showed metallo-beta-lactamase (MBL) production. K. pneumoniae is considered a risk to human health because many of these products do not comply with the microbiological criteria of international and/or Libyan standards. This study emphasized the relationship between K. pneumoniae and raw milk, cheese, milk powder, and infant milk retailed in Libya. There is a need to take the necessary measures to ensure effective hygiene practices during production in dairy factories, handling, and distribution on the market, in particular at a small local production scale.

Clinical features relating to pneumococcal colony phase variation in hospitalized adults with pneumonia.

Background. Streptococcus pneumoniae is a major causative bacteria of pneumonia and invasive pneumococcal disease (IPD); however, the mechanisms underlying its severity and invasion remain to be defined. Pneumococcal colonies exhibit opaque and transparent opacity phase variations, which have been associated with invasive infections and nasal colonization, respectively, in animal studies. This study evaluated the relationship between the opacity of pneumococcal colonies and the clinical presentation of pneumococcal pneumonia.Methods. This retrospective study included adult patients hospitalized with pneumococcal pneumonia between 2012 and 2019 at four tertiary medical institutions. Pneumococcal strains from lower respiratory tract specimens were determined for their serotypes and microscopic colony opacity, and the association between the opacity phase and the severity of pneumonia was evaluated. Serotypes 3 and 37 with mucoid colony phenotypes were excluded from the study because their colony morphologies were clearly different.Results. A total of 92 patients were included. Most patients were older adults (median age: 72 years) and males (67 %), and 59 % had community-acquired pneumonia. Of the 92 patients, 41 (45 %), 12 (13 %), and 39 (42 %) patients had opaque, transparent, and mixed variants in their pneumococcal colony, respectively. The opaque and non-opaque pneumococcal variants had no statistically significant difference in patient backgrounds. Although the pneumonia severity index score did not differ between the opaque and non-opaque groups, the rate of bacteremia was significantly higher in the opaque group than in the non-opaque group. Serotype distribution was similar between the groups.Conclusions. Opaque pneumococcal variants may cause pneumonia and invasive diseases in humans. This study could help elucidate IPD, and opacity assessment may serve as a predictor for IPD.

Phylogenetic analysis of Klebsiella pneumoniae isolates of respiratory tract infections in humans and sheep

Background: Klebsiella pneumoniae is an important opportunistic pathogen, which is capable to colonizing the respiratory system in both human and animals causing mild to severe infections. Aim: This study aims to isolate K. pneumoniae from the nasal discharges of human and sheep as well as identifying the antibiotic resistance and molecular phylogeny of local isolates. Methods: A total of 100; 50 humans and 50 sheep, positive nasal swab isolates were selected, confirmed biochemically and by the VITEK-2 system. Molecular testing using the polymerase chain reaction (PCR) and phylogeny was conducted. Results: On MacConkey agar, Klebsiella isolates were appeared as large, pinkish, and mucoid colonies; while microscopically, it appeared as Gram-negative rods. Traditional biochemical tests revealed that 62% and 78% of human and sheep isolates were positive Klebsiella isolates; whereas respectively, 54.84% and 71.8% of these isolates were positive by VITEK-2. Antibiotic susceptibility test showed that the human isolates were sensitive to aztreonam, piperacillin-tazobactam, ciprofloxacin and cefuroxime. Subsequently, sheep isolates were sensitive to cefuroxime, ciprofloxacin, piperacillin-tazobactam, ampicillin, cefoxitin and tetracycline. Targeting 16S rRNA gene, a total of 17 human and 28 sheep isolates were molecularly positive K. pneumoniae. Phylogenetic analysis of study human and sheep isolates showed its identity to NCBI Indian (LC747146.1) and Iraqi (LC711141.1) isolates, respectively. Comparative analysis between the local human and sheep isolates revealed a significant identity that ranged from 99.82% to 99.88% with a percentage of mutation ranged from 0.008% to 0.002%. Conclusion: Klebsiella pneumoniae is highly prevalent bacterium in both human and sheep with an observable resistance to antibiotics. Molecular phylogeny of study isolates demonstrated their closely relation, suggesting the possible direct or indirect transmission of the bacterium from sheep to human or vice versa. Moreover studies are greatly important to estimate the routes of bacterial transmission. Also, extensive hygiene practices could be lowered the spreading of K. pnuemoniae to farm workers.

Pantoea vagans causing soft rot disease in Agave angustifolia, in Mexico

AbstractIn Mexico, agave (Agave angustifolia Haw.) crops stand out regionally due to their socioeconomic, agroecological and cultural importance. It is the most important raw material in the elaboration of two relevant alcoholic beverages: tequila and mezcal. Nevertheless, this crop is susceptible to phytopathological threats mainly fungi and bacteria, both having a massive impact in yearly production. In this paper, we describe an agave disease consisting in reddish chlorotic lesions along the leaf bases resulting in tissue degradation and producing soft rot (softening tissue and firmness loss). Fast‐growing yellow and mucoid bacteria colonies were isolated from the lesions in BK agar. Microscopic analysis resulted in a Gram‐negative bacilli. Pathogenic tests resulted showed identical symptoms to the initial ones in natural maguey host plants as well as in the selected model Aloe vera L. showing more aggressive soft rot symptoms. It is important to highlight that in the selected plant model (Aloe vera), the symptoms arose faster and more aggressively. Biochemical and molecular analysis of the bacterial isolate identifies it as Pantoea vagans, a species not previously documented in this crop. Although all bacterial infections in plants are treated in similar ways, accurate identification could translate into targeted prevention. The detection of Pantoea vagans is considered a relevant problem given the symptoms it causes. We conclusively reported a 37% infection incidence rate, which can mean significant economic losses. However, the role of the genus Pantoea in agave rot has been poorly studied. Therefore, this report shows the identification of a new Pantoea species bounded to agave cultivars in Malinalco municipality, state of Mexico.

Precise and correct selection of sample – a crucial step in the study of Bacterial Blight Disease (BBD) of pomegranate

Xanthomonas axonopodis PV punicae [Xap] is the causative agent of bacterial blight disease of pomegranate. The disease is severe and can cause upto 80 to 90% reduction in the yield of pomegranate. A variety of approaches have been tried for the control of the disease. The first step in such study is isolation of bacterial blight pathogen Xap from infected pomegranate fruits or leaves and further maintenance of pure culture. Xap develops colourless to faint yellow, highly mucoid colonies on Nutrient Glucose Agar [NGA] medium.. Many researchers get confused at this step with the colonies of Xap. In current research work also, correct isolation method of the pathogen was performed. Here, the suspected Xap pathogen was isolated from the infected plant parts by standard isolation procedure. The suspected pathogen was then streaked on NGA plate. The plate was incubated at 280 C for 72 hr. After incubation, the critical point of study was the selection of Xap colonies. These colonies were selected as suspected Xap isolates. These isolates were purified by repeated subculturing and then preserved on sterile NGA slants. The confirmation of isolate as Xap, causative agent of Bacterial Blight Disease of pomegranate was done by applying Koch’s postulates on healthy plants.

Multidrug Resistance and Molecular Characterization of Klebsiella spp. Isolated from the Cloacal Samples of Broiler Chickens in Bangladesh

Klebsiella spp. poses a significant zoonotic threat, capable of direct and indirect transmission from poultry farms to humans, resulting in severe conditions such as pneumonia, bloodstream infection, enteric fever, meningitis and urinary tract infections. The escalating use of antibiotics in the poultry industry is contributing to the rise of multidrug-resistant (MDR) microorganisms. Thus, a study was undertaken to comprehend the multidrug resistance and virulence gene profile of Klebsiella spp. isolated from poultry cloacal samples sourced from local markets of North Dhaka, Bangladesh. Following aseptic collection, the samples were transported to the laboratory for pure culture isolation. Employing standard microbiological methods such as mucoid colony characteristic, Gram-negative rod-shaped bacteria, biochemical tests and lactose fermentation in selective media, the isolated colonies were presumptively identified. Subsequently, an antibiotic susceptibility test, utilizing the disk diffusion assay, was conducted to assess drug sensitivity in these isolates. A PCR assay was performed to determine the presence or absence of KPC gene of Klebsiella. Among the isolates, ten colonies were identified as Klebsiella spp. based on the colony characteristics, Gram staining, biochemical tests and growth on selective media. Notably, eight isolates (excluding 2 and 4) were incapable of producing indole from tryptophan, indicating the presence of two different types of species. All isolates exhibited resistance to at least three antibiotics, namely ceftazidime, ampicillin and cefoxitin. Isolate 2 and 5 were demonstrated additional resistance to azithromycin, while isolate 9 and 10 exhibited further resistance to streptomycin and doxycycline. Molecular analysis indicated that 40% of these isolates harbor KPC virulent gene (880bp), with the most multidrug resistant isolate 9 and 10 possessing this gene. This study is pivotal for assessing the prevalence of virulent and MDR Klebsiella spp. in chicken cloacal samples, providing insights into shedding and transmission risk on soil and water environment, as well as to humans.

Variovorax durovernensis sp. nov., a novel species isolated from an infected prosthetic aortic graft in a human.

A novel bacterial strain, GSTT-20T was isolated from an infected, prosthetic endovascular graft explanted from a shepherd in London, United Kingdom. This strain was an aerobic, catalase-positive, oxidase-negative, Gram-stain-negative, motile, curved rod. It grew on blood agar, chocolate agar and MacConkey agar incubated at 37 °C in an aerobic environment after 48 h, appearing as yellow, mucoid colonies. Analysis of the complete 16S rRNA gene sequence showed closest similarity to Variovorax paradoxus with 99.6 % identity and Variovorax boronicumulans with 99.5 % identity. Phylogenetic analysis of the 16S rRNA gene sequence and phylogenomic analysis of single nucleotide polymorphisms within 1530 core genes showed GSTT-20T forms a distinct lineage in the genus Variovorax of the family Comamonadaceae. In silico DNA-DNA hybridization assays against GSTT-20T were estimated at 32.1 % for V. boronicumulans and 31.9 % for V. paradoxus. Genome similarity based on average nucleotide identity was 87.50 % when comparing GSTT-20T to V. paradoxus. Based on these results, the strain represented a novel species for which the name Variovorax durovernensis sp. nov. was proposed. The type strain is GSTT-20T (NCTC 14621T=CECT 30390T).

Phage selection drives resistance-virulence trade-offs in Ralstonia solanacearum plant-pathogenic bacterium irrespective of the growth temperature.

While temperature has been shown to affect the survival and growth of bacteria and their phage parasites, it is unclear if trade-offs between phage resistance and other bacterial traits depend on the temperature. Here, we experimentally compared the evolution of phage resistance-virulence trade-offs and underlying molecular mechanisms in phytopathogenic Ralstonia solanacearum bacterium at 25 °C and 35 °C temperature environments. We found that while phages reduced R. solanacearum densities relatively more at 25 °C, no difference in the final level of phage resistance was observed between temperature treatments. Instead, small colony variants (SCVs) with increased growth rate and mutations in the quorum-sensing (QS) signaling receptor gene, phcS, evolved in both temperature treatments. Interestingly, SCVs were also phage-resistant and reached higher frequencies in the presence of phages. Evolving phage resistance was costly, resulting in reduced carrying capacity, biofilm formation, and virulence in planta, possibly due to loss of QS-mediated expression of key virulence genes. We also observed mucoid phage-resistant colonies that showed loss of virulence and reduced twitching motility likely due to parallel mutations in prepilin peptidase gene, pilD. Moreover, phage-resistant SCVs from 35 °C-phage treatment had parallel mutations in type II secretion system (T2SS) genes (gspE and gspF). Adsorption assays confirmed the role of pilD as a phage receptor, while no loss of adsorption was found with phcS or T2SS mutants, indicative of other downstream phage resistance mechanisms. Additional transcriptomic analysis revealed upregulation of CBASS and type I restriction-modification phage defense systems in response to phage exposure, which coincided with reduced expression of motility and virulence-associated genes, including pilD and type II and III secretion systems. Together, these results suggest that while phage resistance-virulence trade-offs are not affected by the growth temperature, they could be mediated through both pre- and postinfection phage resistance mechanisms.

Persistent ciprofloxacin exposure induced the transformation of Klebsiella pneumoniae small colony variant into mucous phenotype

IntroductionSmall colony variant (SCV) is a bacterial phenotype closely related to persistent and recurrent infections. SCVs are mutations that occur within bacterial populations, resulting in a change in bacterial morphology and the formation of small colonies. This morphological change may enhance bacterial resistance to antibiotics and contribute to persistent and recurrent infections.MethodsWe isolated Klebsiella pneumoniae (KPN) and its SCV from a child with recurrent respiratory tract infections. KPN and SCV were treated with subinhibitory concentrations of antibiotics. growth curves, serum resistance experiments, macrophage phagocytosis experiments and whole genome sequencing were used to characterize KPN and SCV.ResultsAfter treating KPN and SCV with subinhibitory concentrations of antibiotics, we found that ciprofloxacin induced the SCV transition to the mucoid phenotype. We found that the growth of mucoid Klebsiella pneumoniae was significantly slower than maternal strain and SCV though growth curves. Serum resistance experiments showed that mucoid strains had significantly higher serum resistance compared to maternal strain and SCV. Macrophage phagocytosis experiments revealed that SCV had significantly higher intracellular survival rates compared to maternal strain and mucoid strains. Differential gene analysis of three strains revealed that the mucoid strain contained DNA polymerase V subunit UmuC gene on the plasmid, while the SCV strain had an additional IcmK family IV secretion protein on its plasmid.DiscussionOur study showed the SCV of KPN changed to a mucoid colony when exposed to subinhibitory concentrations of ciprofloxacin. The higher resistance of serum of mucoid colonies was possibly related to the UmuC gene, while the increased intracellular survival of SCV may be related to the IcmK family type IV secretion proteins.

Cystic fibrosis and pulmonary biofilms

Cystic fibrosis (CF) is an autosomal recessive disorder that involves the cystic fibrosis transmembrane conductance regulator. This protein is an anion channel that transfers chloride and bicarbonate from an intracellular location to an extracellular location. This transfer supports the formation of a normal periciliary fluid layer that is essential for ciliary function and the clearance of particulates from bronchi. Changes in mucociliary function can result in chronic bronchitis and recurrent infections. Studies using micro computed tomography of explanted lungs from CF patients have demonstrated that there is a significant reduction in the number of terminal bronchi and that approximately 50% of these bronchi are obstructed with secretions. These airways become colonized with bacteria, such as Pseudomonas aeruginosa, which can form biofilms, and this results in chronic inflammation and chronic airway damage. Biofilm formation can be predicted when mucoid colonies of pseudomonas grow out on routine bacterial cultures of sputum. Studies on the prognosis of patients with CF have demonstrated that there is lack of clear genotype-phenotype correlation, but one recent longitudinal study did report an association between more severe CFTR genotypes and clinical outcomes. In addition, the degree of pulmonary dysfunction and pseudomonas colonization, especially with drug resistant pseudomonas, predict poor outcomes. Bacteria in biofilms are more resistant to antibiotics, and the structure of biofilms limits host defense responses. Treatment approaches include the use of more than one antibiotic, the addition of inhaled antibiotics, the use of enzymes that degrade biofilms, and the use of small molecules that inhibit community metabolism in biofilms.&#x0D; &#x0D; Key words: cystic fibrosis transmembrane conductance regulator, extracellular secretions, periciliary fluid, Pseudomonas aeruginosa, biofilms

First Report of Fire Blight on Chinese hawthorn (Crataegus pinnatifida Bunge) Caused by Erwinia amylovora in Korea.

Fire blight is one of the destructive plant diseases caused by Erwinia amylovora and causes enormous economic losses worldwide. Fire blight was initially reported in apples, pears, and Chinese quince (Park et al. 2016; Myung et al. 2016a, 2016b) in Korea, but recent studies have reported new hosts such as apricot (Lee et al. 2021) and mountain ash (Lim et al, 2023). These reports indicate that fire blight is likely to disperse to new hosts in Korea. During the nationwide survey in June 2021, we observed typical symptoms of blossom blight and shoot blight on a Chinese hawthorn (Crataegus pinnatifida Bunge) just near an orchard (37°09'21.7"N, 127°35'02.6"E) in Icheon, Gyeonggi Province, where fire blight of Asian pear occurred. For identifying its causal agent, bacterial isolates were recovered after incubating at 28 ℃ for 24 hours on tryptic soy agar (TSA) medium (BD Difco, USA) from blighted leaves and shoots that were surface sterilized with 70% alcohol for 30 sec and homogenized in 500 µl of 10mM MgCl2. Pure cultures of white to mucoid colonies were grown on mannitol glutamate yeast extract (MGY) medium, a semi-selective medium for E. amylovora (Shrestha et al, 2003). Two isolates produced 1.5 kb amplicon through colony PCR using amsB primers (Bereswill et al. 1995). Two strains (CPFB26 and CPFB27) from the Chinese hawthorn produced amplicons identical to that from the TS3128 strain of E. amylovora, isolated from the pear tree and identified in 2016 (Park et al. 2016). For the partial 16s rRNA sequences, the total DNA of these two strains was extracted using the Wizard DNA prep kit (Promega, USA), and PCR was performed using fD1 (5'-AGAGTTTGATCCTGGCTCAG-3') and Rp2 (5'-ACGGCTACCTTGTTACGACTT-3') primer sets and further sequenced (Weisburg et al. 1991). These sequences belonged to the E. amylovora clade and were identified as E. amylovora in phylogenetic analysis (GenBank accession no. OP753569 and OP753570). Based on BLASTN analysis, CPFB26 and CPFB27 showed 99.78% similarity to the sequences of the E. amylovora strains TS3128, CFBP 1430, and ATCC 49946. To confirm pathogenicity of the isolates, 10 ㎕ bacterial suspensions (1.5 ⅹ 108 CFU/ml) was injected through the veins of the upper 2nd leaf of 3-month-old clone of apple rootstock (Malus domestica cv. M29) and incubated for six days at 28 ℃ in a chamber with 12 hours of light per day. Petioles and stems turned red hue, and the shoots finally blighted. To complete Koch's postulates, colonies were recovered on TSA medium from the inoculated apple rootstocks and verified through colony PCR for the amsB and A/B primer set (Powney et al. 2011). Hawthorn has been reported as an epidemiologically important alternate host plant of fire blight (van der Zwet et al. 2012). This study is the first to report fire blight caused by E. amylovora in Chinese hawthorn in Korea. Because Chinese hawthorn is natively distributed in Korea and is widely used as a landscaping tree (Jang et al. 2006), the findings of this study suggest that early monitoring could prevent the spread of fire blight through natural hosts.

Effect of Chemical Mutagenesis on Salt Tolerance of Common Beans (Phaseolus vulgaris L.) Nodulating Rhizobium

The chemical mutation is one of the important factors in the enrichment of the salt resistance capacity of the rhizobium isolates. Therefore, this study focused on 10 isolates of rhizobium collected from soil samples of Babile, Hararghe region, Ethiopia. All the collected isolates turned into a moderately yellow, yellow, and deep yellow color in yeast extract mannitol agar medium (YEMA) containing bromothymol blue after 48 h of incubation. It indicated that all the isolates were acid-producing Rhizobia. Moreover, based on colony morphology and diameter, 70% of the isolates displayed large mucoid colonies, and 30% of the isolates showed large watery colonies in YEMA media. Among the 10 isolates, 9(90%), 9(90%), 6(60%), 6(60%), 2(20%), and 1(10%), were grown at different salinity levels such as 2%, 4%, 6%, 8%, 9% and 10% of NaCl, correspondingly. The most salt-resistant wild isolate was HUCR 6 collected from Babile soil grown at 10% NaCl salinity level. A total of six mutants were considered after chemical mutagenesis based on their capacity to survive in the extreme salinity levels (11 to 14%). Mutant isolates such as HUCRM 4, HUCRM 5, and HUCRM 6 were the most tolerant Rhizobium that grew at the salinity level of 11 to 14% NaCl. The most sensitive mutant isolate was HUCRM 10 followed by the isolates HUCRM 8 and HUCRM 9 was the next sensitive mutant Rhizobia that grew only at 11% of NaCl concentration. Compared to the Rhizobium wild isolates, the mutant isolates were observed to be more tolerant to a medium containing higher concentration of NaCl, as high as 11% to 14%. Besides, 80% of the mutant isolates demonstrated effective nodulation with the common beans. The mutant isolate (HUCRM 4) showed better performance in relation to root nodule performance of Rhizobium species and increased the plant biomass production. In this study, mutant isolates HUCRM 6, which is tolerated to 14% NaCl, and HUCRM 4, HUCRM 5, and HUCRM 6 isolates tolerated at 12% salinity level. Finally, based on their symbiotic efficiency and tolerance to extreme salt levels, these mutant isolates (HUCRM 4, HUCRM 5 and HUCRM 6) were encouraged to be used for the development of Rhizobium inoculants of common beans grown under extreme saline conditions. J. Bio-Sci. 30(2): 13-22, 2022

The enigmatical manipulators in the capsule synthesis of Pasteurella multocida: Iron acquisition proteins

Aim: Spontaneous capsular loss or capsular changes in Pasteurella multocida can result from repeated laboratory passages, positive or negative regulatory genes, or an unknown gene. This study, it was aimed to compare the properties of phenotypic, genotypic, and biotypic of each non-typical, and typical Pasteu-rella multocida strain, to determine the dominant genes on capsule synthesis. Materials and Methods: Fifty-six strains, which capsular type was determined, and thirty -six, which capsular type was not determined, were used in this study. Multiple linear regression analysis was used based on the presence of dominant genes (serogroup, serotype, toxin, adhesin, iron acquisition, and protectin) in the isolates. Results: When colony morphologies of strains were evaluated of these strains by culture methods, mucoid colony formation was commonly detected in typical strains (87.5%), in contrast to non-typical strains (27.7%). In typical strains, the highest percentages of ptfA, ompA, and tadD genes were 78.57%, 75%, and 69.64%, respectively. In non-typical strains, the highest rates of ompA, ptfA, and tadD genes were 61.1%, 52.78%, and 52.78%, respectively. According to multiple linear regression analysis results, the together hgbA with hgbB genes caused the increase of capsule synthesis in these strains. The presence of the ompA gene in these strains was secondly a induction on these strains. Other genes were not effective in capsule synthesis in these strains. Conclusion: It was determined that the most significant effect in the forming of non-typical P. multocida strains was related to not enough HgbA and HgbB genes. It was supposed that P. multocida may not be heavily encapsulated under iron-restricted conditions. Consequently, P. multocida may change its capsule or lose its capsule related to iron acquisition proteins.

Association between phenotypic characteristics of Escherichia coli and UTI recurrence in immunocompromised patients: A case-control study

Urinary tract infection (UTI) recurrence is important in immunocompromised patients. There is a trend to study genotypically and phenotypically the role of certain virulence factors of Escherichia coli in the diagnosis of recurrent UTI. The main objective of this study was to determine if there is an association between phenotypic characteristics of E coli and UTI recurrence in immunocompromised patients. A case-control study was performed on immunocompromised patients from Hospital Regional de Alta Especialidad del Bajío, Mexico. E coli strains isolated from these patients were identificated and antimicrobial susceptibility test were performed. Strains with filamented cell morphology, mucoid colonial phenotype, or biofilm production were considered cases. Strains without the characteristics were considered controls. UTI recurrence was identified based on clinical records. The odds ratio (OR) was calculated to quantify the magnitude of the association. An association between filamented cell morphology and UTI recurrence was found (OR=2.19 95% CI 1.06-4.51; P=.031). No association was found between mucoid colony morphology (P>.05) or biofilm production (P>.05) and UTI recurrence. An association between mucoid colony morphology and extended-spectrum β-lactamase production was found (OR=3.09 95% 1.59-5.99; P<.001). Filamented cell morphology and mucoid colonial phenotype may have a possible diagnostic value for the detection of UTI recurrence and antimicrobial resistance. Further diagnostic test studies are needed to fully assess their clinical utility.