Plasmid Profiles Research Articles

Novel plasmids in multidrug-resistant Shigella flexneri serotypes from Pakistan

Shigellosis is the main cause of food and waterborne diarrhea and is an emerging threat to human health. The current study characterized the indigenous multidrug-resistant Shigella flexneri serotypes for their plasmid profiles and genetic diversity, to characterize the plasmid evolutionary patterns and distribution. In total, 199 identified S. flexneri isolates belonging to six different serotypes were analyzed for plasmid profiling, followed by an analysis of whole genome sequencing. All isolates of S. flexneri resistant to antibiotics harbored multiple copies of plasmids with sizes ranging from 1.25 kbp to 9.4kbp. These isolates were clustered into 22 distinct plasmid patterns, labeled as p1-p22. Among these, p1 (24%) and p10 (13%) were the predominant plasmid profiles. All S. flexneri strains were grouped into 12 clades with a 75% similarity level. Also, a significant association was observed among the plasmid patterns, p23 and p17 with the drug-resistant patterns AMC, SXT, C (19.5%) and OFX, AMC, NA, CIP (13.5%), respectively. Moreover, the most widespread plasmid patterns p4, p10, and p1 showed a significant association with the serotypes 1b (29.16%), 2b (36%), and 7a (100%), respectively. After plasmid sequence assembly and annotation analysis, a variety of small plasmids that vary in size from 973 to 6200bp were discovered. Many of these plasmids displayed high homology and coverage with plasmids from non-S. flexneri. Several novel plasmids of small size were discovered in multidrug-resistant S. flexneri. The data also showed that plasmid profile analysis is more consistent than antibiotic susceptibility pattern analysis for identifying epidemic strains of S. flexneri isolated in Pakistan.

Whole-Genome Sequencing and Comparative Genomics Analysis of a Newly Emerged Multidrug-Resistant Klebsiella pneumoniae Isolate of ST967.

Klebsiella pneumoniae is a common cause of hospital- and community-acquired infections globally, yet its population structure remains unknown for many regions, particularly in low- and middle-income countries (LMICs). Here, we report for the first-time whole-genome sequencing (WGS) of a multidrug-resistant K. pneumoniae isolate, ARM01, recovered from a patient in Armenia. Antibiotic susceptibility testing revealed that ARM01 was resistant to ampicillin, amoxicillin-clavulanic acid, ceftazidime, cefepime, norfloxacin, levofloxacin, and chloramphenicol. Genome sequencing analysis revealed that ARM01 belonged to sequence type 967 (ST967), capsule type K18, and antigen type O1. ARM01 carried 13 antimicrobial resistance (AMR) genes, including blaSHV-27, dfrA12, tet(A), sul1, sul2, catII.2, mphA, qnrS1, aadA2, aph3-Ia, strA, and strB and the extended-spectrum β-lactamase (ESBL) gene blaCTX-M-15, but only one known virulence factor gene, yagZ/ecpA, and one plasmid replicon, IncFIB(K)(pCAV1099-114), were detected. The plasmid profile, AMR genes, virulence factors, accessory gene profile, and evolutionary analyses of ARM01 showed high similarity to isolates recovered from Qatar (SRR11267909 and SRR11267906). The date of the most recent common ancestor (MRCA) of ARM01 was estimated to be around 2017 (95% confidence interval [CI], 2017 to 2018). Although in this study, we report the comparative genomics analysis of only one isolate, it emphasizes the importance of genomic surveillance for emerging pathogens, urging the need for implementation of more effective infection prevention and control practices. IMPORTANCE Whole-genome sequencing and population genetics analysis of K. pneumoniae are scarce from LMICs, and none has been reported for Armenia. Multilevel comparative analysis revealed that ARM01 (an isolate belonging to a newly emerged K. pneumoniae ST967 lineage) was genetically similar to two isolates recovered from Qatar. ARM01 was resistant to a wide range of antibiotics, reflecting the unregulated usage of antibiotics (in most LMICs, antibiotic use is typically unregulated.) Understanding the genetic makeup of these newly emerging lineages will aid in optimizing antibiotic use for patient treatment and contribute to the worldwide efforts of pathogen and AMR surveillance and implementation of more effective infection prevention and control strategies.

Profiling and antibiotic resistance of some human intestinal isolates

The cases of antibiotic resistance in Enterobacteriaceae are of great global concern. This study aimed at contributing towards the fight against antibiotic resistance and ameliorate the management/treatment of Enterobacteriaceae-linked diseases. Ten (10) rectal swab samples were collected from five (5) male and five (5) females and two isolates were identified from the samples collected. The isolates were identified through colonial, morphological and biochemical tests carried out following standard procedures. Isolates were investigated for their antibiotic resistance profile, Multiple Antibiotic Resistance indices (MARi), pathogenicity and their resistance genes were identified through molecular means using plasmid amplification and primers. Primers used include. ermB, BlaTem, qnrB genes Result obtained showed the isolates to be Escherichia coli and Klebsiella oxytoca. E. coli showed Alpha (α) heamolysis, while Klebsiella oxytoca showed gamma (γ) heamolysis . E. coli was resistant to 75% of the antibiotics used, Klebsiella oxytoca was sensitive to 42% of all the antibiotics. All the test organisms were resistant to all classes of Cephalosporins. The plasmid profiling revealed that all isolates have low molecular weight plasmids. The molecular fingerprinting of the isolates using gene primers viz -a viz ermB, BlaTem, qnrB genes showed E. coli to have resistance genes for macrolides (ermB gene) while none of the isolates had resistance factor against quinolones (qnrB gene). This study showed a high carriage of Enterobacteriaceae having phenotypic resistance with corresponding plasmid-borne resistance genes. The need to understand how bacteria adapt to the antibiotic environment will lead to new therapeutic strategies for antibiotic-resistant infections. Interventions measures to minimize the abundance of antibiotic-resistant commensals and opportunistic pathogens may include faecal microbiota transplantation and the use of live biotherapeutics.

Characterization of Shigella flexneri Serotype 6 Strains Isolated from Bangladesh and Identification of a New Phylogenetic Cluster.

A significant cause of shigellosis in Bangladesh and other developing countries is Shigella flexneri serotype 6. This serotype has been subtyped, on the basis of the absence or presence of a group-specific antigen, E1037, into S. flexneri 6a and 6b, respectively. Here, we provided rationales for the subclassification, using several phenotypic and molecular tools. A set of S. flexneri 6a and 6b strains isolated between 1997 and 2015 were characterized by analyzing their biochemical properties, plasmid profiles, virulence markers, pulsed-field gel electrophoresis (PFGE) results, and ribotype. Additionally, the genomic relatedness of these subserotypes was investigated with global isolates of serotype 6 using publicly available genomes. Both subserotypes of S. flexneri 6 agglutinated with monoclonal antiserum against S. flexneri (MASF) B and type VI-specific antiserum (MASF VI) and were PCR positive for O-antigen flippase-specific genes and virulence markers (ipaH, ial, sen, and sigA). Unlike S. flexneri 6a strains, S. flexneri 6b strains seroagglutinated with anti-E1037 antibodies, MASF IV-I. Notably, these two antigenically distinct subserotypes were clonally diverse, showing two distinct PFGE patterns following the digestion of chromosomal DNA with either XbaI or IceuI. In addition, hybridization of a 16S rRNA gene probe with HindIII-digested genomic DNA yielded two distinguishing ribotypes. Genomic comparison of S. flexneri subserotype 6a and 6b strains from Bangladesh indicated that, although these strains were in genomic synteny, the majority of them formed a unique phylogroup (PG-4) that was missing for the global isolates. This study supports the subserotyping and emphasizes the need for global monitoring of the S. flexneri subserotypes 6a and 6b. IMPORTANCE Shigella flexneri serotype 6 is one of the predominant serotypes among shigellosis cases in Bangladesh. Characterization of a novel subserotype of S. flexneri 6 (VI:E1037), agglutinated with type 6-specific antibody and anti-E1037, indicates a unique evolutionary ancestry. PFGE genotyping supports the finding that these two antigenically distinct subserotypes are clonally diverse. A phylogenetic study based on single-nucleotide polymorphism (SNP) data revealed that these two subserotypes were in genomic synteny, although their genomes were reduced. Interestingly, a majority of the S. flexneri 6 strains isolated from Bangladesh form a novel phylogenetic cluster. Therefore, this report underpins the global monitoring and tracking of the novel subserotype.

Status of MDR and Plasmid Profiling of ESBL producing E. coli and Klebsiella spp. isolated from milk of Bovine in Gangetic Plain Zone of Uttar Pradesh

Nowadays resistance to antibiotics of β-lactam groups is expanding rapidly and is one of the latest challenges faced by scientific community due to therapeutic failure. In the present study total 180 milk samples were collected from two district of Gangetic Plain Zone of Uttar Pradesh (India). Total 65 (36.11%) isolates were confirmed comprising 44 (24.4%) E. coli and 21 (11.67%) Klebsiella spp. by PCR analysis using uidA gene and 16S rRNA gene respectively, 35/180 (19.44%) isolates were confirmed as ESBL producers, constituting 12 (6.67%) E. coli and 23 (12.8%) Klebsiella spp. All ESBL positive isolates were subjected to antimicrobial resistance pattern using 17 antibiotics of 10 different classes. In this study all the isolates of E. coli and Klebsiella spp. were found 100% resistant to cefotaxime, cefpodoxime, ceftriazone and ampicillin. Both the isolates of E. coli and Klebsiella spp. revealed different susceptibility pattern to different non-β-lactam antibiotics except aminoglycosides class of antibiotics for which both the isolates were found 100% sensitive. The multi-drug resistance pattern was assessed by using MAR index. Out of total ESBL producers, 80.0% isolates were found MDR with 78.0% E. coli and 83.0% Klebsiella spp. Overall 12 resistance pattern were observed for E. coli ranging from 5 to 12 antibiotics and 09 for Klebsiella spp. ranging from 5 to 14 antibiotics. Plasmid profiling revealed total 71.42% isolates carrying plasmids with average molecular weight ranged between 1.0 Kb to > 10 Kb and number1-4.

Emergence of OXA-484-Producing Klebsiella variicola in China.

The frequent and inappropriate use of antibiotics has caused a dramatic rise in the number, species, and degree of multi-drug resistant bacteria, making them more prevalent and difficult to treat. In this context, the aim of the present study was to characterize the OXA-484-producing strains isolated from a perianal swab of a patient by using whole-genome analysis. In this study, carbapenemase-producing Klebsiella variicola was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), average nucleotide identity (ANI) and PCR. S1 nuclease pulsed-field gel electrophoresis (S1-PFGE) and Southern blotting were utilized to characterize the plasmid profiles of K. variicola 4717. In particular, WGS was performed to obtain genomic information on this clinical isolate, and assemble all the plasmids of the bla OXA-484-harboring strain. The antimicrobial susceptibility pattern of K. variicola 4717 revealed that it was resistant to a range of antibiotics, including aztreonam, imipenem, meropenem, ceftriaxone, cefotaxime, ceftazidime, levofloxacin, ciprofloxacin, piperacillin-tazobactam, methylene-sulfamer oxazole, amoxicillin-clavulanic acid, cefepime, and tigecycline. Its susceptibility to chloromycin was intermediate, while it was still susceptible to amikacin, gentamicin, fosfomycin, and polymyxin B. The presence of two companion plasmids, p4717_1 and p4717_2, together with a plasmid carrying the bla OXA-484 gene was observed. An in-depth investigation of p4717-OXA-484 uncovered that it is an IncX3-type plasmid and shares a similar segment encoded by IS26. Given the similar genetic background, it was conceivable that bla OXA-484 could have developed from bla OXA-181 through a series of mutations. Herein, we described the first genome sequence of K. variicola strain harbouring the class D β-actamase bla OXA-484 in an Inc-X3-type plasmid. Our work also uncovered the genetic characterization of K. variicola 4717 and the importance of initiating antimicrobial detection promptly.

Extensively drug-resistant Acinetobacter baumannii: role of conjugative plasmids in transferring resistance.

Acinetobacter baumannii is one of the most successful pathogens that can cause difficult-to-treat nosocomial infections. Outbreaks and infections caused by multi-drug resistant A. baumannii are prevalent worldwide, with only a few antibiotics are currently available for treatments. Plasmids represent an ideal vehicle for acquiring and transferring resistance genes in A. baumannii. Five extensively drug-resistant A. baumannii clinical isolates from three major Jordanian hospitals were fully sequenced. Whole-Genome Sequences (WGS) were used to study the antimicrobial resistance and virulence genes, sequence types, and phylogenetic relationship of the isolates. Plasmids were characterized In-silico, followed by conjugation, and plasmid curing experiments. Eight plasmids were recovered; resistance plasmids carrying either aminoglycosides or sulfonamide genes were detected. Chromosomal resistance genes included blaOXA-66, blaOXA-91, and blaOXA-23, and the detected virulence factors were involved in biofilm formation, adhesion, and many other mechanisms. Conjugation and plasmid curing experiments resulted in the transfer or loss of several resistance phenotypes. Plasmid profiling along with phylogenetic analyses revealed high similarities between two A. baumannii isolates recovered from two different intensive care units (ICU). The high similarities between the isolates of the study, especially the two ICU isolates, suggest that there is a common A. baumannii strain prevailing in different ICU wards in Jordanian hospitals. Three resistance genes were plasmid-borne, and the transfer of the resistance phenotype emphasizes the role and importance of conjugative plasmids in spreading resistance among A. baumannii clinical strains.

In vitro Antibacterial Potency of Leaf Extract of Moringa oleifera against NFGNB Isolated from UTI Patients and their Plasmid Profiling

Non-fermenting gram-negative bacteria (NFGNB) endeavouring as major pathogen in infectious disease, predominantly in urinary tract infection (UTI) and increased resistance in NFGNB are matter of concern. This study aimed to evaluate the frequency of NFGNB, antibiotics resistance pattern, plasmid profiling, and antibacterial efficacy of Moringa oleifera against NFGNB. NFGNB were isolated from clinically suspected UTI patients. Identification of isolates and their antibiotics sensitivity pattern were analyzed according to conventional method, and Vitek 2 automated system. Moreover, NFGNB were evaluated for biofilm production and presence of plasmid. Furthermore, antibacterial activity of Moringa oleifera was evaluated against NFGNB. P. aeruginosa (86.0%), and A. baumannii (10.0%) were the most frequent NFGNB followed by Providencia rettgeri 2.0%, Stenotrophomonas maltophilia 1.0%, myroides species 1.0%. 68.6% P. aeruginosa and 60.0% A. baumannii were biofilm producers whereas imipenem and meropenem were the most effective antibiotics. Isolated NFGNB showed multiple bands of plasmid. Furthermore, Moringa oleifera leaves extract showed antibacterial activity against tested NFGNB. MDR-NFGNB presents challenges in treatment and Moringa oleifera leaf extract may be used as an alternative medicine. However, the therapeutic role of specific ingredients present in extract needs further investigation and purification.

The rise of multidrug resistant Salmonella isolates in healthy chickens in Brazil by successful establishment of plasmid IncHI2A carrying several antibiotic resistance genes.

Salmonella spp. is an important global issue in food-producing animals. The present study evaluated antimicrobial resistance and virulence profiles in Salmonella spp. isolates from chickens in Brazil. Identification of serotypes, virulence and antimicrobial resistance genes, and plasmid profiles were performed. Three different serovars were found, S. Schwarzengrund, S. Newport and S. Kentucky. All isolates were considered Multidrug- resistance (MDR). Among the 32 Salmonella spp. isolates analysed, 29 isolates carried blaCTX-M-2 gene and showed the insertion sequence ISCR1 and a class 1 integron structure upstream from blaCTX-M-2. This gene was harboured in large IncHI2A plasmids with approximately 280kb. Furthermore, 30 isolates harboured tetA and tetB genes and 25 also harboured qnrB. The virulence genes invA, misL, orfL, spiC and pipD were detected in all isolates. The study shows a high prevalence of MDR Salmonella isolates disseminated in poultry farms. The association of the replicon IncHI2A with the resistance genes found, elevate the risk of foodborne disease outbreaks.

Plasmid Profile and Enterobacterial Repetitive Intergenic Consensus-PCR Characterization of Salmonella Infantis Isolates Recovered From Poultry Sources

Background: Salmonella is known as one of the most important bacterial agents infecting both humans and animals. Salmonella Infantis has been reported as one of the 15 most prevalent serovars worldwide. Despite its clinical importance, there is little information on the molecular characteristics of S. Infantis in Iran. Objectives: This study was conducted to characterize S. Infantis isolates collected from poultry sources in the last decade. The isolates were typified by plasmid profile and enterobacterial repetitive intergenic consensus (ERIC-PCR). Methods: Forty S. Infantis isolates from poultry sources were subjected to plasmid profile and ERIC-PCR characterization. We used a commercial plasmid extraction kit to extract and purify plasmid DNA which then was separated by gel electrophoresis and viewed under a UV transilluminator. For ERIC-PCR, a commercial bacterial chromosomal DNA extraction kit was used. In this study, we chose ERIC2 primer for the ERIC-PCR test. Results: The plasmid profile revealed that 35% of isolates did not contain any plasmids, but the rest (65%) carried a variable number of plasmids with different molecular weights. Six plasmid profiles were found among 40 S. Infantis isolates. Using ERIC2 primer, 7 profiles were found among 40 S. Infantis isolates in ERIC-PCR. Bands with molecular weights ranging from 400 to 3000 bp were observed. Conclusion: This study provided some genetic data on S. Infantis isolates recovered from poultry sources, and these data can be used for a broader epidemiological study nationwide. These findings showed that although plasmid and ERIC profiles are valuable in epidemiological studies, they have some limitations, too.

Prevalence, antimicrobial susceptibility, and antibiotic resistance gene transfer of Bacillus strains isolated from pasteurized milk.

Pasteurization is carried out in dairy industries to kill harmful bacteria present in raw milk. However, endospore-forming bacteria, such as Bacillus, cannot be completely eliminated by pasteurization. In this study, a total of 114 Bacillus strains were isolated from 133 pasteurized milk samples. Antibiotic susceptibility tests showed that the percentage of Bacillus with intrinsic resistance to ampicillin and penicillin were 80 and 86%, respectively. Meanwhile, some Bacillus isolates had acquired resistance, including trimethoprim-sulfamethoxazole resistance (10 isolates), clindamycin resistance (8 isolates), erythromycin resistance (2 isolates), and tetracycline resistance (1 isolate). To further locate these acquired resistance genes, the plasmids were investigated in these 16 Bacillus strains. The plasmid profile indicated that Bacillus cereus BA008, BA117, and BA119 harbored plasmids, respectively. Subsequently, the Illumina Novaseq PE150 was applied for the genomic and plasmid DNA sequencing. Notably, the gene tetL encoding tetracycline efflux protein was found to be located on plasmid pBC46-TL of B. cereus BA117. In vitro conjugative transfer indicated that pBC46-TL can be transferred into Bacillus invictae BA142, Bacillus safensis BA143, and Bacillus licheniformis BA130. The frequencies were of 1.5 × 10-7 to 1.7 × 10-5 transconjugants per donor cells. Therefore, Bacillus strains with acquired antibiotic resistance may represent a potential risk for the spread of antibiotic resistance between Bacillus and other clinical pathogens via horizontal gene transfer.

Pheno- and genotypic characteristics of Yersinia pestis strains from phylogroups 2.MED4 and 2.MED1 – etiological agents of 20th-century plague outbreaks in the Caspian Sea region

ABSTRACT We conducted a comparative analysis of the phenotypic and genotypic characteristics of plague pathogen strains from the 2.MED4 and 2.MED1 phylogroups, representing the main subspecies of the medieval Yersinia pestis biovar found in the plague foci of the Caspian Sea region, which were epidemically active in the 20th century. We evaluated the biochemical properties, nutritional requirements, and plasmid profiles of Y. pestis strains within the 2.MED4 and 2.MED1 phylogroups of the medieval biovar. We discovered the variability in the virulence-associated genes hemS, caf1M, and ssaJ in the strains of the 2.MED lineage. Our data on the phenotypic and genotypic characteristics of the medieval biovar can be used in laboratory diagnostics of the plague microbe and contribute to the understanding of genomic changes during the microevolution of this highly virulent biovar.

Prevalence and Plasmid Profiles of Enterobacteriaceae Isolates Obtained from Individuals with Asymptomatic Bacteriuria in Okada, Edo State, Nigeria

Asymptomatic bacteriuria (ASB) is the presence of bacteria in apparently healthy individuals. Although, the prevalence varies widely with age, gender, and the presence or absence of genitourinary abnormalities, the detection of pathogenic bacteria in urine constitutes firm evidence of infection. The objectives of this study were to isolate members of the family Enterobacteriaceae from urine samples of research participants, and to determine the antibacterial susceptibility and plasmid profiles of the isolates. A total of 122 mid-stream urine samples consisting of 72 male and 50 female participants from selected primary and secondary schools were collected. The age of the participants used in this study ranged between 4 years to 19 years old. Isolates were cultured on MacConkey agar and Cysteine lactose electrolyte deficient agar and then incubated for 24 hr at 37 °C. The overnight culture was then subjected to antibiotic susceptibility testing using the disk diffusion test as described by Kirby-Bauer. Thereafter, plasmid analysis was carried out. The statistical package used to analyze the samples was IBM SPSS v23 to cover the calculations for the chi-square, student t-test, and analysis of variance. The frequency of bacterial isolates with respect to age were obtained, as follows: 4–8 (9.0%), 9–13 (16.4%), 14–18 (2.5%) and >19 (1.6%). The prevalence of the four (23; 63.9%) Enterobacteriaceae isolate groups were: Escherichia coli (36.1%), Citrobacter species (11.1%), Klebsiella species (8.3%) and Enterobacter species (8.3%) while non Enterobacteriaceae (13) was 36.1%. A prevalence of 25.9% of ASB was reported in this study where the percentage occurrence of bacteria isolates showed high prevalence in the male participants, at 17.2% compared to the 12.3% reported for the female participants. The antibiogram showed that all isolates were susceptible to Cefprozil except isolates belonging Klebsiella species that showed resistance to the antibiotic. Citrobacter species and Klebsiella species isolates were susceptible to Ofloxacin while Enterobacter species isolates was only susceptible to Cefixime. The percentage figures from resistant plasmid profile analysis indicated that Cefuroxime, Cefprozil and Ofloxacin showed relatively good susceptibility profile against the nine bacterial isolates that were subjected to the analysis. Resistance After plasmid curing was majorly chromosomally-borne. From this research, the prevalence of ASB was found yo be high in Okada, with the infection being higher in males having Escherichia coli as the commonest bacterial isolate. Hence, there is need for early routine evaluation as well as detection of this silent infection for prompt referral and intervention.

Emergence of mcr-8.2-harboring hypervirulent ST412 Klebsiella pneumoniae strain from pediatric sepsis: A comparative genomic survey

ABSTRACT Emerging mobile colistin resistance (mcr) genes pose a significant threat to public health for colistin was used as the last resort to treat multidrug-resistant (MDR) pathogenic bacterial infections. Hypervirulent Klebsiella pneumoniae (hvKP) is a clinically significant pathogen resulting in highly invasive infections, often complicated by devastating dissemination. Worryingly, the untreatable and severe infections caused by mcr-harbouring hvKP leave the selection of antibiotics for clinical anti-infective treatment in a dilemma. Herein, we screened 3,461 isolates from a tertiary teaching hospital from November 2018 to March 2021, and an mcr-8.2-harbouring hvKP FAHZZU2591 with a conjugative plasmid was identified from paediatric sepsis. This is the first report of MCR-8-producing hvKP from paediatric sepsis to our best knowledge. The susceptibility, genetic features, and plasmid profiles of the isolate were investigated. Further, we assessed the virulence potential of FAHZZU2591 and verified its pathogenicity and invasive capacity using a mouse model. The phylogenetic analysis of mcr-8-bearing K. pneumoniae revealed that China is the predominant reservoir of the mcr-8 gene, and the clinic is the primary source. Our work highlights the risk for the spread of mcr-positive hvKP in clinical, especially in paediatric sepsis, and the persistent surveillance of colistin-resistance hvKP is urgent.

Bacteriological assessments of indoor air in the staff quarters of a tertiary institution in Benin City, Nigeria

Indoor air contains large number of airborne microorganisms such as bacteria and fungi and their estimation is important for use as index of cleanliness for any particular environment and to determine the relation they bear on human health. This study was aimed at determining the bacteriological air quality of the living rooms in some selected Staff Quarters in University of Benin and University of Benin Teaching Hospital. The airborne bacterial loads of indoor air in the living rooms of Twelve (12) Staff Quarters in University of Benin and University of Benin Teaching Hospital both in Benin City were determined using the Settle Plate methods. The houses were categorized as Apartment 1 which represents University of Benin Junior Staff Quarters (JSQ), Apartment 2 which represents University of Benin Teaching Hospital Quarters, and Apartment 3 which represents University of Benin Senior Staff Quarters (SSQ). The living rooms were sampled twice a month between April, 2017 and September, 2017. The airborne samples were studied using standard microbiological methods and Polymerase Chain Reaction and 16S rRNA techniques were used for the gene sequencing. The antibiotic susceptibility pattern and plasmid profile of the characterized airborne bacterial isolates were evaluated using spread plate and agarose gel electrophoresis methods. The temperature and relative humidity of the indoor air environment in the sampled areas in the living rooms were determined using the thermometer and hygrometer respectively. The mean indoor temperature and relative humidity for Apartments 1, 2 and 3 ranged from 26.10 ± 1.190C to 31.20 ± 0.870C and 81 ± 2.70% to 87 ± 1.89% respectively. The mean indoor airborne bacterial counts in Apartment 1 and Apartment 3 ranged between 1.00 x 103 ± 0cfu/m3 to 4.42 x 103 ± 1.42cfu/m3 and 1.09 x 103 ± 0.09cfu/m3 to 5.17 x 103 ± 3.17cfu/m3 respectively. In Apartment 2, the counts ranged from 1.00 x 103 ± 0.00cfu/m3 to 6.99 x 103 ± 4.69cfu/m3. The difference in the airborne bacterial counts obtained in the morning and afternoon period of study in Apartment 1 was statistically significant (P<0.05) while Apartments 2 and 3 showed no significant differences respectively (P>0.05). Ten airborne bacterial isolates were characterized, further characterization by molecular techniques, confirmed them to be identified as Staphylococcus aureus strain S33 R, Bacillus subtilis subsp. strain 168, Oceanobacillus manasiensis strain YD3-56, Streptomyces vietnamensis strain GIM4.0001, Actinosynnema pretiosum strain C-15003, Micrococcus caseolyticus strain 235, Ornithinibacillus composti strain GSS05 and Bacillus ectoiniformans strain NE-14. General and personal hygienic practices are important for healthy living, and living rooms used for close contact interactions and good ventilation.

Genotype classification of Moraxella bovis using MALDI-TOF MS profiles.

Moraxella bovis (M. bovis) is regarded as a causative agent of infectious bovine keratoconjunctivitis (IBK), the most common ocular disease of cattle. Recently, whole genome sequencing identified the presence of two distinct genotypes within M. bovis that differ in chromosome content, potential virulence factors, as well as prophage and plasmid profiles. It is unclear if the genotypes equally associate with IBK or if one is more likely to be isolated from IBK lesions. We utilized 39 strains of M. bovis that had previously undergone whole genome sequencing and genotype classification to determine the utility of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) to accurately genotype M. bovis strains. We successfully developed two biomarker models that accurately classified strains according to genotype with an overall accuracy of 85.8-100% depending upon the model and sample preparation method used. These models provide a practical tool to enable studies of genotype associations with disease, allow for epidemiological studies at the sub-species level, and can be used to enhance disease prevention strategies.

Analysis of plasmid profiles of Escherichia coli bacteria and their resistance to several antibiotics

Escherichia coli is a bacteria belonging to the family Enterobacteriaceae. Escherichia coli is a harmful gram bacterium living in the digestive tract. These bacteria also live in water and soil, commonly referred to as coliform. This research aims to analyze the plasmid profile of Escherichia coli bacteria and its resistance to some antibiotics. To explore the plasmid profiles of Escherichia coli in a recent research electrophoresis gel, the antibiotic used in its resistance tests is the Amoxicillin, Tetracycline antibiotic, Ciprofloxacin, Sulfamethoxazole, and Streptomycin antibiotic. The electrophoresis results in a difference in the number of plasmid profiles with 1 to 2 ribbons of plasmid DNA different sizes from ˜1500 bp to ˜2300 bp. Antibiotic resistance tests have occurred in Amoxicillin, Ciprofloxacin, and some resistance samples to the Streptomycin antibiotic

The Genetic Characteristics and Carbapenem Resistance Mechanism of ST307 Klebsiella pneumoniae Coharbouring blaCMY-6, blaOXA-48, and a Truncated blaNDM-1.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a common nosocomial pathogen causing severe infectious diseases, and ST307 CRKP is an emerging clone. In this study, we collected five ST307 CRKP isolates, evaluated their antimicrobial susceptibility using microbroth dilution, and their clonality and population structure by PFGE, cgMLST, and SNP-based phylogenetic analysis. Then, the genome characteristics, such as antimicrobial resistance genes and plasmid profiles, were studied by subsequent genomic analysis. The plasmid transfer ability was evaluated by conjugation, and the carbapenem resistance mechanism was elucidated by gene cloning. The results showed that all five ST307 CRKP isolates harboured blaCMY-6, blaOXA-48, and blaNDM-1; however, the end of the blaNDM-1 signal peptide was interrupted and truncated by an IS10 element, resulting in the deactivation of carbapenemase. The ST307 isolates were closely related, and belonged to the globally disseminated clade. blaOXA-48 and blaNDM-1 were located on the different mobilisable IncL/M- and IncA/C2-type plasmids, respectively, and either the pOXA-48 or pNDM-1 transconjugants were ertapenem resistant. Gene cloning showed that blaCMY-6 could elevate the MICs of carbapenems up to 64-fold and was located on the same plasmid as blaNDM-1. In summary, ST307 is a high-risk clone type, and its prevalence should be given additional attention.

Whole genome sequencing of Moraxella bovis strains from North America reveals two genotypes with different genetic determinants

BackgroundMoraxella bovis and Moraxella bovoculi both associate with infectious bovine keratoconjunctivitis (IBK), an economically significant and painful ocular disease that affects cattle worldwide. There are two genotypes of M. bovoculi (genotypes 1 and 2) that differ in their gene content and potential virulence factors, although neither have been experimentally shown to cause IBK. M. bovis is a causative IBK agent, however, not all strains carry a complete assortment of known virulence factors. The goals of this study were to determine the population structure and depth of M. bovis genomic diversity, and to compare core and accessory genes and predicted outer membrane protein profiles both within and between M. bovis and M. bovoculi.ResultsPhylogenetic trees and bioinformatic analyses of 36 M. bovis chromosomes sequenced in this study and additional available chromosomes of M. bovis and both genotype 1 and 2 M. bovoculi, showed there are two genotypes (1 and 2) of M. bovis. The two M. bovis genotypes share a core of 2015 genes, with 121 and 186 genes specific to genotype 1 and 2, respectively. The two genotypes differ by their chromosome size and prophage content, encoded protein variants of the virulence factor hemolysin, and by their affiliation with different plasmids. Eight plasmid types were identified in this study, with types 1 and 6 observed in 88 and 56% of genotype 2 strains, respectively, and absent from genotype 1 strains. Only type 1 plasmids contained one or two gene copies encoding filamentous haemagglutinin-like proteins potentially involved with adhesion. A core of 1403 genes was shared between the genotype 1 and 2 strains of both M. bovis and M. bovoculi, which encoded a total of nine predicted outer membrane proteins.ConclusionsThere are two genotypes of M. bovis that differ in both chromosome content and plasmid profiles and thus may not equally associate with IBK. Immunological reagents specifically targeting select genotypes of M. bovis, or all genotypes of M. bovis and M. bovoculi together could be designed from the outer membrane proteins identified in this study.

Multidrug Resistance Tracing by Plasmid Profile Analysis and the Curing of Bacteria from Different Clinical Specimens

Human-pathogenic bacteria resistant to one or multiple antibiotics have dramatically increased worldwide in the past decades. These bacteria possess great danger, have become a global issue, and it is now impossible to avoid developing strategies for the restoration of treatment options against infections caused by them. This research aims at profiling plasmids of multidrug-resistant bacteria from various clinical specimens such as ear exudate, sputum, urethral swab, wound swab, urine from the catheter, urine, nasal swab, high vaginal swab, stool, eye swab, and blood at Chukwuemeka Odumegwu Ojukwu University Teaching Hospital, Awka, Anambra State, Nigeria. Our investigation used the agar diffusion method for susceptibility tests and identification of multidrug-resistant bacteria before plasmid extraction and gel electrophoresis. A plasmid curing test was performed with 10% sodium dodecyl sulphate. Of the 860 bacteria whose resistance profile was determined, only 42 were multidrug-resistant. These bacteria include Pseudomonas aeruginosa 16 (38.10%), Staphylococcus aureus 12 (28.57%), Escherichia coli 9 (21.43%), and Klebsiella pneumoniae 5(11.90%). The molecular weight of their plasmids ranges between 20.884 kbp and 187.50 kbp. As indicated by the plasmid bands, some bacteria had similar molecular weight while others had no plasmid. The bacterial pattern of the postcuring sensitivity test showed that the bacteria with plasmid bands were cured as they became susceptible to the drugs they were previously resistant to, while the bacteria without plasmid bands remained resistant to the antibiotics. This implies that the latter’s multidrug resistance is nonplasmid mediated. Our analyses highlight the relationship between plasmids and multidrug resistance as well as the role of plasmids in the transmission of drug resistance across bacteria. Thus, in order to lessen the burden that multidrug-resistant bacteria cause and to improve bacterial infections treatments, there should be continued surveillance and periodic research on antibiotic resistance patterns of bacteria from various clinical settings.