Pneumonia Isolates Research Articles

CC180 clade dynamics does not universally explain Streptococcus pneumoniae serotype 3 persistence post-vaccine: a global comparative population genomics study.

Background Clonal complex 180 (CC180) is currently the major clone of serotype 3 Streptococcus pneumoniae (Spn). The 13-valent pneumococcal conjugate vaccine (PCV13) does not have significant efficacy against serotype 3 despite polysaccharide inclusion in the vaccine. It was hypothesized that PCV13 may effectively control Clade I of CC180 but that Clades III and IV are resistant, provoking a population shift that enables serotype 3 persistence. This has been observed in the United States, England, and Wales but not Spain. We tested this hypothesis further utilizing a dataset from Portugal. Methods We whole-genome sequenced (WGS) 501 serotype 3 strains from Portugal isolated from patients with pneumococcal infections between 1999-2020. The draft genomes underwent phylogenetic analyses, pangenome profiling, and a genome-wide association study (GWAS). We also completed antibiotic susceptibility testing and compiled over 2,600 serotype 3 multilocus sequence type 180 (MLST180) WGSs to perform global comparative genomics. Findings CC180 Clades I, II, III, IV, and VI distributions were similar when comparing non-invasive pneumonia isolates and invasive disease isolates (Fisher's exact test, P=0.29), and adult and pediatric cases (Fisher's exact test, P=0.074). The serotype 3 CCs shifted post-PCV13 (Fisher's exact test, P<0.0001) and Clade I became dominant. Clade I is largely antibiotic-sensitive and carries the phiOXC141 prophage but the pangenome is heterogenous. Strains from Portugal and Spain, where Clade I remains dominant post-PCV13, have larger pangenomes and are associated with the presence of two genes encoding hypothetical proteins. Interpretation Clade I became dominant in Portugal post-PCV13, despite the burden of the prophage and antibiotic sensitivity. The accessory genome content may mitigate these fitness costs. Regional differences in Clade I prevalence and pangenome heterogeneity suggest that clade dynamics is not a generalizable approach to understanding serotype 3 vaccine escape. Funding National Institute of Child Health and Human Development, Pfizer, and Merck Sharp & Dohme.

Antibiofilm Activity of Silver Nanoparticles Synthesized from Seed Extract of Garcinia Kola

Silver nanoparticles from plant extracts are novel compounds with potential antimicrobial properties. Studies on antibiofilm activity of Ag-NPs synthesized from seed extracts of Garcinnia kola (G. kola) were carried out. Garcinnia kola seed were obtained from Keffi market, Nigeria. Green synthesis of Ag-NPs from the seed was carried using 2.0mm silver-nitrate by use of standard method. The Ag-NPs synthesized from the seed were characterized using former transmission infrared (FITR) spectroscopy and scanning election microscope. The antimicrobial activity of the Ag-NPs against Klebsiella pneumonia (Kp) isolates were carried out using agar dilution method. The biofilm formation by the isolates as well as the inhibition and dissolution by Ag-NPs were eval__uated using microplate method. The functional groups detected in the Ag-NPs were N-H, C-O, N-O, and CΞC with peaks 906.5cm-1,1282.2cm-2, 13344cm-1, 1550.6cm-1 and 217.1cm-1 respectively. The size of the particles ranges from 179-296nm. The minimum inhibiting concentration (MICs) of the particles and meropenem against the isolates were 250µg/l and 4.0µg/l. The functional inhibiting concentrates of the particles were 1.0. The optical clarity of biofilm formed by the isolates was 2.073 and 2.049. the percentage biofilm inhibiting effects of the particles was highest apart. KpC (K. Pneumoniae ATCC BAA 1075) with percentage inhibit ranges from 27.28-21.67% at 80-12.5% of the MICs. The percentage inhibiting effect of Ag-NPs in with meropenem was highest at MICs but low in MIC 12.5 with percentage inhibition 28.26% and 27.18%. The Ag-NPs alone and antibacterial activity and biofilm inhibiting effect while Ag-NPs in with meropenem had effect but against isolate but with potential antibiofilm activity.

Comparison of Mutant Prevention Concentrations of Fluoroquinolones Against ESBL-Positive and ESBL-Negative Klebsiella pneumoniae Isolates from Orthopedic Patients.

The majority of Klebsiella pneumonia isolates possess the extended-spectrum beta-lactamase (ESBL) enzymes. Therefore, K. pneumoniae can easily develop drug resistance. How to effectively overcome the problem of drug resistance in K. pneumoniae is still a research hotspot. This study aimed to compare the mutant prevention concentration (MPC) of ESBL-positive and ESBL-negative K. pneumoniae isolated from orthopedic patients, which may provide a basis for the effective use of drugs to control the enrichment of resistance mutants of K. pneumoniae. The MPC90 values of 55 isolates of ESBL-positive K. pneumoniae against 4 fluoroquinolones were 32 µg/mL for levofloxacin and gatifloxacin, 16 µg/mL for ciprofloxacin, and 4 µg/mL for gemifloxacin. The selection index value was 8 for levofloxacin and ciprofloxacin and 2 for gemifloxacin and gatifloxacin, respectively. For ESBL-negative K. pneumoniae isolates, the MPC90 values were 16 µg/mL for levofloxacin and ciprofloxacin, 4 µg/mL for gemifloxacin, and 32 µg/mL for gatifloxacin. The selection index value was 8 for levofloxacin and ciprofloxacin, 2 for gemifloxacin, and 4 for gatifloxacin. For the ESBL-positive K. pneumoniae, the %T>MIC90 order was gemifloxacin > levofloxacin > ciprofloxacin > gatifloxacin. For the ESBL-negative K. pneumoniae, the %T>MIC90 order was levofloxacin > gemifloxacin > ciprofloxacin > gatifloxacin. The mutant-preventing ability of gatifloxacin and gemifloxacin was the strongest among the 4 fluoroquinolones. So gemifloxacin may be the first choice of drug to treat K. pneumoniae infection.

Genetic Characterization of Extended-Spectrum Beta-Lactamase (ESBL) and Metallo-Beta-Lactamase (MBL) Producing Klebsiella pneumoniae from Diabetic Foot Ulcer (DFU)

ABSTRACT Background: Antibiotic resistance in common pathogenic bacteria is linked with the genetic makeup. The genetic basis of antibiotic resistance may vary in different species or pathophysiological conditions. Objectives: We studied the antibiotic resistance in Klebsiella pneumonia isolates from DFU in the western Indian population. We also studied the presence of ESBL and MBL mechanisms of antibiotic resistance along with the prevalence of the genes involved in ESBL (TEM ESBL , SHV ESBL , and CTX-M ESBL ) and MBL (NDM-1 bla , KPC bla , OXA-48 bla , and VIM bla ) production. Results: A total of 161 K. pneumoniae isolates were analyzed; among which 50.93% were positive for ESBL and 45.96% were positive for MBL production. Most of the isolates were resistant to antibiotics used in the present study and partially resistant to Imipenem and Amikacin. There was no relation between the antibiotic resistance of the isolates and the production of ESBL or MBL mechanism of antibiotic resistance. Further, TEM ESBL was the most prevalent gene in K. pneumoniae isolates followed by CTX-M ESBL , NDM-1 bla , SHV ESBL , and KPC bla . VIM bla was the least prevalent gene found in K. pneumoniae isolates. There was no difference in the prevalence of the genes with respect to the presence or absence of ESBL and MBL mechanism of resistance. Further, there was no relation between the prevalence of the genes and antibiotic resistance in K. pneumoniae isolates. Conclusion: These results along with the literature review suggest that the prevalence of the genes involved in antibiotic resistance mechanisms are widespread in India and their distribution varies in different studies.

Epidemiological, Virulence, and Antibiotic Resistance Analysis of Klebsiella pneumoniae, a Major Source of Threat to Livestock and Poultry in Some Regions of Xinjiang, China.

Klebsiella pneumoniae (K. pneumoniae) is recognized as a zoonotic pathogen with an increasing threat to livestock and poultry. However, research on K. pneumoniae of animal origin remains limited. To address the gap, a comprehensive investigation was carried out by collecting a total of 311 samples from the farms of four animal species (dairy cow, chicken, sheep, and pig) in selected areas of Xinjiang, China. Isolates were identified by khe gene amplification and 16S rRNA gene sequencing. Genotyping of K. pneumonia isolates was performed using wzi typing and multilocus sequence typing (MLST). PCR was employed to identify virulence and resistance genes. An antibiotic susceptibility test was conducted using the Kirby-Bauer method. The findings revealed an isolation of 62 K. pneumoniae strains, with an average isolation rate of 19.94%, with the highest proportion originating from cattle sources (33.33%). Over 85.00% of these isolates harbored six virulence genes (wabG, uge, fimH, markD, entB, and ureA); while more than 75.00% of isolates possessed four resistance genes (blaTEM, blaSHV, oqxA, and gyrA). All isolates exhibited complete resistance to ampicillin and demonstrated substantial resistance to sulfisoxazole, amoxicillin/clavulanic acid, and enrofloxacin, with an antibiotic resistance rate of more than 50%. Furthermore, 48.39% (30/62) of isolates were classified as multidrug-resistant (MDR) strains, with a significantly higher isolation rate observed in the swine farms (66.67%) compared to other farms. Genetic characterization revealed the classification of the 62 isolates into 30 distinct wzi allele types or 35 different sequence types (STs). Notably, we identified K. pneumoniae strains of dairy and swine origin belonging to the same ST42 and wzi33-KL64 types, as well as strains of dairy and chicken origin belonging to the same wzi31-KL31-K31 type. These findings emphasize the widespread occurrence of drug-resistant K. pneumoniae across diverse animal sources in Xinjiang, underscoring the high prevalence of multidrug resistance. Additionally, our results suggest the potential for animal-to-animal transmission of K. pneumoniae and there was a correlation between virulence genes and antibiotic resistance genes. Moreover, the current study provides valuable data on the prevalence, antibiotic resistance, and genetic diversity of K. pneumoniae originating from diverse animal sources in Xinjiang, China.

Antibiotic Susceptibility of Klebsiella pneumonia Isolates from Hospitalized Patients in Three Referral Medical Centers in Isfahan, Iran

: Background: Klebsiella pneumoniae (K. pneumoniae) is a major cause of nosocomial and community-acquired infections (CAIs). Understanding the antibiotic susceptibility of this bacterium is essential for selecting the most effective antibiotic for associated infections. Objectives: We investigated the sensitivity of K. pneumonia (KP) isolates obtained from inpatients in Isfahan, Iran. Methods: Organism identification and antibiotic susceptibility testing were conducted using standard tests. Stratification was performed based on extended spectrum beta-lactamase (ESBL) production and the source of infection acquisition (community versus hospital). Results: A total of 437 KP isolates were investigated, of which 5.5% were pan drug resistant (PDR), 56.8% were extensively drug resistant (XDR), and 18% were ESBL producers. The isolates were most susceptible to colistin (100%), aminoglycosides (62.8%), and carbapenems (55%). Nosocomial isolates showed lower sensitivity to most of the examined antibiotics. Conclusions: The high prevalence of PDR and XDR KP strains in hospitalized patients is a major problem in the studied area.

Bacterial profile, antimicrobial susceptibility pattern and associated risk factors of urinary tract infection among pregnant women attending tertiary care hospital in Rawalpindi

The study objective to enhance treatment effectiveness by assessing risk factors, bacterial profiles, and antimicrobial susceptibility of uropathogens in pregnant women. During the period from March 2023 to August 2023, we examined 210 urine samples obtained frompregnant women. The samples were subjected to uropathogens isolation, identification and susceptibility testing against thirteen antibiotic classes using the Kirby Bauer disk diffusion method, following standard procedures, to identify uropathogens in cases of significant bacteriuria. In our study there is no significant link found between age, educational level, socioeconomic status, and parity in relation to UTI during pregnancy. In 42% of the samples with significant bacteriuria, Escherichia coli emerged as the predominant uropathogens, followed by Pseudomonas aeruginosa, Staphylococcus saprophyticus, and Klebsiella pneumoniae, in that order. E. coli displayed the greatest resistance to ciprofloxacin and ceftazidime, yet demonstrated high susceptibility to meropenem, imipenem, fosfomycin,and amikacin. Furthermore, a significant portion of K. pneumonia isolates exhibited resistance to ceftazidime and ciprofloxacin. For S. saprophytic, resistance was observed against penicillin and gentamicin, although they displayed 100% sensitivity to vancomycin and 81.2% sensitivity to Nitrofurantoin. The current study findings revealed that factors influencing pregnant women's UTI risk, include age, literacy, residence, multigravida, multipara, infection history, and trimester status. Access to clean water sources also played a role, highlighting the importance of addressing these variables in maternal health toreduce UTI incidence. Screening antenatal patients for UTIs is imperative, and it is advisable to conduct regular screenings for pregnant women. This practice helps monitor uropathogens sensitivity patterns and facilitates the development of antibiotic protocols based on local susceptibility data.

DETECTION OF OQXAB EFFLUX PUMPS GENE IN ESCHERICHIA COLI AND KLEBSIELLA PNEUMONIA ISOLATED FROM PREGNANT WOMEN SUFFERED FROM URINARY TRACT INFECTIONS AND DIABETES MELLITUS IN BASRAH

Considerably, pregnant women with diabetes mellitus are highly susceptible to urinary tract infections. The aim of the study is investigate the incidence of OqxAB efflux pumps gene among Escherichia coli and Klebsiella pneumonia isolates from urine of pregnant women suffered from urinary tract infections and Diabetes mellitus. E.coli and K.pneumonia were identified using conventional methods and confirmed by specific 16S rDNA primers. Antibiotic susceptibility test of E. coli and K. pneumonia isolates against several antibiotics were examined using Vitek®-2 compact system .The production of efflux pump by E. coli and K. pneumonia isolates were investigated phenotypically using ethidium bromide-agar cartwheel method (EtBr CW). Moreover, OqxAB Efflux Pumps genes were tested. The results showed that among 100 urine samples, 40 urine samples showed positive bacterial growth where 21(51%) E coli isolates, 8(20%) K. pneumonia isolates. In terms of antibiotic sensitivity, all E.coli and K.pneumonia isolates were resistant to ampicillin, piperacillin and ticarcillin, respectively. The prevalence of OqxAB efflux pump genes frequency ranges from 72% in E. coli and 50% in K. pneumonia.

Two novel phages, Klebsiella phage GADU21 and Escherichia phage GADU22, from the urine samples of patients with urinary tract infection.

Phages are found in a wide variety of places where bacteria exist including body fluids. The aim of the present study was to isolate phages from the urine samples of patients with urinary tract infection. The 10 urine samples were cultured to isolate bacteria and also used as phage sources against the isolated bacteria. From 10 urine samples with positive cultures, 3 phages were isolated (33%) and two of them were further studied. The Klebsiella phage GADU21 and Escherichia phage GADU22 phages infected Klebsiella pneumonia and Escherichia coli, respectively. Among the tested 14 species for host range analysis, the Klebsiella phage GADU21 was able to infect two species which are Klebsiella pneumonia and Proteus mirabilis, and Escherichia phage GADU22 was able to infect four species which are Shigella flexneri, Shigella sonnei and Escherichia coli. Among different isolates of the indicator bacteria for each phage, GADU21 infected half of the tested 20 Klebsiella pneumonia isolates while GADU22 infected 85% of the tested 20 E. coli isolates. The genome sizes and GC ratios were 75,968bp and 44.4%, and 168,023bp and 35.3% for GADU21 and GADU22, respectively. GADU21 and GADU22 were both lytic and had no antibiotic resistance and virulence genes. GADU21 was homologue with Klebsiella phage vB_KpP_FBKp27 but only 88% of the genome was covered by this phage. The non-covered parts of the GADU21 genome included genes for tail-fiber-proteins and HNH-endonuclease. GADU22 had 94.8% homology with Escherichia phage vB_Eco_OMNI12 and had genes for immunity proteins. Phylogenetic analysis showed GADU21 and GADU22 were members of Schitoviridae family and Efbeekayvirus genus and Straboviridae family and Tevenvirinae genus, respectively. VIRIDIC analysis classified these phages in new species clusters. Our study demonstrated the possibility to use infected body fluids as phage sources to isolate novel phages. GADU21 is the first reported Klebsiella phage isolated from human body fluid. The absence of virulence and antibiotic resistance genes in their genomes makes the phages a potential therapeutic tool against infections.

Investigation of Antimicrobial Susceptibility Patterns and blaVIM -metallo-β-lactamase Gene in Clinical Samples of Klebsiella pneumoniae

Multidrug resistance is a widespread issue that plays an important role in disease outcome. This study was designed to isolate Klebsiella pneumonia in different clinical specimens and detected their Antibiotic resistance profile. A total of 319 samples were collected from various clinical specimens for both genders and different ages. The samples were streaked on the blood and MacConkey agars. The bacterial growth identified using biochemical tests and Vitek®2 systems to confirm it. Also, the Vitek®2 system was used to detect the antibiotic sensitivity. Out of 319 clinical samples, K. pneumonia was identified in 67 (21%) cases. The highest isolation rate was in urine 25(37.3%), followed by sputum 13(19.4%), and the least isolation was in CSF with one isolate (1.5%). The results revealed that K. pneumonia isolates were multi-drug resistant pathogens (MDR) with high resistance to Ampicillin (97%) and 85% for piperacillin. The PCR results revealed blaVIM- genes frequency was 20 (30%). K. pneumonia is one of the bacteria that cause urinary tract infections, and it is a widespread multidrug-resistant pathogen, and blaVIM- producing K. pneumoniae are found in clinical samples at Thi-Qar hospitals. Therefore, monitoring the administration of antibiotics and their rational use is necessary to reduce antimicrobial resistance and treatment failure.

EMERGENCE OF CEFTAZIDIME-AVIBACTAM RESISTANCE IN ENTERO-BACTERALES AND PSEUDOMONAS AERUGINOSA

Objective: The objective of this study is to determine the emergence of resistance to Ceftazidime-Avibactam (CAZ-AVI) by Enterobacterales and Pseudomonas aeruginosa in clinical isolates. Material and Methods: In this cross-sectional study (6-months) March-August 2022 Carbapenem resistant Enterobacterales were tested for Ceftazidime-Avibactam (30/20 µg, Oxoid Pvt Ltd) using Disk diffusion technique and enzymes were identified in resistant strains by Carbapenem Inactivation Method (mCIM, eCIM) as per CLSI M100 Guidelines 2022. Results: CAZ-AVI effectiveness has greatly decreased among Enterobacterales and P. aeruginosa isolates in recent past. Antimicrobial susceptibility testing results were interpreted using CLSI M100 document. Resistance against CAZ-AVI in Enterobacterales was found to be 80.8 % in E. coli and 72.1% in Klebsiella pneumonia isolates. This higher emergence is associated with CRE isolates majorly comprising MBLs in our country. Moreover, it has been observed that Metallo- β-lactamases mediated enzyme resistance is one of the major resistance patterns followed by serine carbapenemases. Conclusion: The high frequency of resistance 77% was observed against CAZ-AVI in CRE and in CRPA, the resistance is 80.1% respectively. In our country this tremendously increase in CAZ-AVI resistance is attributed to the existence of NDM in the region. Keywords: Carbapenem Resistant Enterobacterales (CRE), Metallo beta lactamases (MBLs), Carbapenem Resistant Pseudomonas aeruginosa (CRPA) Ceftazidime-Avibactam (CAZ-AVI), Modified Carbapenem Inactivation Method (mCIM), EDTA-modified Carbapenem Inactivation Method(eCIM), Multiple Drug Resistance (MDR)

2170. It takes two to tango: ß-lactam/ß-lactamase inhibitor combinations targeting metallo-ß-lactamase (MBL)- producing Klebsiella pneumoniae.

Abstract Background The emergence of pan drug resistant (PDR) gram-negatives co-producing NDM-1 and CTX-M-15 calls for devising more efficient biocidal regimens. Also, understanding the salient transcriptomic and phenotypic changes that contribute to persistence which aid in pathogen survival is critical to therapeutic preparation in the clinical setting. Methods A 10-day hollow fiber infection model (HFIM) was utilized to simulate humanized β-lactam regimens involving imipenem (IMI) 1g q8h, aztreonam (ATM) 2g q8h, and ceftazidime/avibactam (CAZ/AVI) 2g/500mg alone and in 3-drug combination regimens against a PDR Klebsiella pneumonia isolate co-harboring blaNDM-1 and blaCMY-6, blaCTX-M-15 and blaSHV-2. Static time kills were performed to investigate the interplay between IMI and ATM. At 2h post drug exposure, total RNA was analyzed by qRT-PCR for the expression of pertinent genes. Glutaraldehyde-fixed samples were imaged using fluorescence and scanning electron microscopy. Results In HFIM, IMI contributed synergistically to the antimicrobial action as the average bacterial concentration between 30-120h was 2.1 log10 CFU/mL lower in ATM + CAZ/AVI + IMI than in ATM + CAZ/AVI (2.44 log10 CFU/mL vs. 4.54 log10 CFU/mL, respectively). Adding IMI to ATM+ CAZ/AVI also resulted in an enhanced post-antibiotic effect. Although ATM + IMI reduced bacterial counts by ≥4 log10 CFU/mL in 6h time kill assays, regrowth to the system carrying capacity of ∼8.5 log10 CFU/mL by 24h in all arms rendered these drugs ineffective. By 2h of treatment, ATM alone resulted in long filamentous cells whereas IMI resulted in significant populations of spheroplasts. ATM+IMI caused blebbing in the center of elongating filaments followed by a burst in spheroplast formation. qRT-PCR showed upregulation of spheroplast protein Y (spy) and penicillin binding proteins (pbp1, pbp2, pbp3) for all combinations involving IMI. However, pbp1, pbp3 and spy remained unchanged and pbp2 levels were downregulated by >25% in ATM monotherapy. Conclusion Overall, this PDR clinical isolate persisted in the presence of ATM + CAZ/AVI. The addition of IMI to target PBP2 in β-lactam combinations maximally suppressed the emergence of filamentous persisters and may be a promising strategy to prepare for increasing rates of gram-negative resistance. Disclosures All Authors: No reported disclosures

In vitro activity of novel apramycin-dextran nanoparticles and free apramycin against selected Dutch and Pakistani Klebsiella pneumonia isolates

Klebsiella pneumoniae are bacteria associated with respiratory tract infections and are increasingly becoming resistant to antibiotics, including carbapenems. Apramycin is a veterinary antibiotic that may have the potential to be re-purposed for use in human health, for example, for the treatment of respiratory tract infections after coupling to inhalable nanoparticles. In the present study, the antibiotic apramycin was formulated with single chain polymeric nanoparticles and tested in free and formulated forms against a set of 13 Klebsiella pneumoniae isolates (from the Netherlands and Pakistan) expressing different aminoglycoside resistance phenotypes. Minimum Inhibitory Concentration, Time Kill Kinetics and biofilm experiments were performed providing evidence for the potential efficacy of apramycin and apramycin-based nanomedicines for the treatment of human Klebsiella pneumonia infections.

Gut acquisition of Extended-spectrum β-lactamases-producing Klebsiella pneumoniae in preterm neonates: Critical role of enteral feeding, and endotracheal tubes in the neonatal intensive care unit (NICU).

Klebsiella spp. can colonize the intestine of preterm neonates, and over-growth has been associated with necrotizing enterocolitis, hospital-acquired infections, and late-onset sepsis. This could lead us to suggest that the clinical pertinence of intestinal colonization with ESBL in preterm neonates appears to be important. We conducted this study to characterize the genetic proprieties of ESBL-producing Klebsiella pneumoniae (ESBL-KP) under clinical isolates and to describe the risk factors for the intestinal tract acquisition event during hospitalization. One hundred and thirteen premature infants were recruited from the neonatal intensive care unit (NICU). All newborns are issued from the birth suites of the pregnancy department. Two rectal swabs were planned to define K. Pneumoniae intestinal carriage status. ESBL-KP was confirmed by Brilliance ESBL selective chromogenic Agar. Antimicrobial susceptibility testing including phenotypic testing and genotypic detection of the most commonly described ESBL genes was done. Logistic regression models were performed to find the variables associated with the acquisition event of ESBL-KP. A total of 62 (54.86%) premature neonates were colonized with ESBL-KP. The rate of blaSHV, blaTEM, blaCTX-M1, blaCTX-M2, blaCTX-M9, and blaOXA-48 genes among the isolates was 82, 48, 93.5, 4.8, 11.2 and 3.22%, respectively. We found that ESBLs K. Pneumoniae isolates were 100% resistant to amoxicillin, clavulanic acid-amoxicillin, cefotaxime, ceftazidime, and gentamicin. The regression model is for a given significant association between the tract intestinal of ESBL-KP acquisition events and the use of enteral tube feeding (OR = 38.46, 95% CI: 7.86-188.20, p-Value: 0.001), and endotracheal tubes (OR = 4.86, 95% CI: 1.37-17.19, p-Value 0.014). Our finding supposes that the enteral feeding tube and endotracheal tube might have a critical role in colonizing the intestinal tract of preterm infants. This highlights the current status of both practices that will require updated procedures in the NICU.

Detection of Biofilm Formation Among the Clinical Isolates of Klebsiella pneumoniae: Phenotypic and Genotypic Methods

Infections caused by biofilm-embedded pathogens decrease the efficacy of traditional treatments and increase antibiotic tolerance. Most of the human bacterial infections are biofilm-associated. Therefore, this study aimed to detect the biofilm formation among the clinical isolates of Klebsiella pneumonia collected from different hospitals in Wasit province-Iraq by phenotypic and genotypic methods. 525 clinical samples were used to isolate 77 K. pneumoniae strains from clinical specimens for five months. They were identified by microbiological method as K. pneumoniae. The microtiter plate method is used to detect the biofilm formation. Results showed that out of 77 K. pneumonia isolates, 76 (98.7%) isolates were biofilm producers with three different categories; 12 (15.6%) were weak-biofilm producers, while other isolates 63 (81.8%) and 1 (1.3%) were moderate and vigorous producers, respectively. However, 1 (1.3%) isolates were identified as nonbiofilm producers. Amplification of genes by multiplex PCR technique was done for 77 isolates of K. pneumonia to detect biofilm production genes, mrkD and FimH. Results showed that out of 77 isolates, there were 74 isolates (94.8%) positive to mrkD and 33 isolates (42.8%) to fimH. Keywords: K. pneumonia; Microtiter plate method; mrkD; fimH; Iraq.

EVALUATION OF QUORUM SENSING SIGNALS OF STRONG BIOFILM PRODUCING BACTERIA VIA LC-MSMS, HPLC AND BIOSENSORS

This study aimed to show presence of Quorum Sensing (QS) signals of Gram-negative and Gram-positive biofilm producing bacteria isolated from real dairy process lines. Defining the profile and chemical composition of QS-signals is an important factor in control of microbial resistance and biofilm production. We especially focused on unusual behaviour of Gram-positive and Gram-negative isolates. Long-chain acyl-homoserine lactones (AHLs) signals (C14-HSL, C16-HSL and C18-HSL) and DFD (4,5-dihidroksi-2,3-pentanedione)-AI-2 signals of the isolates were studied by High-performance liquid chromatography (HPLC) and Liquid Chromatography with tandem mass spectrometry (LC-MSMS) methods. All Gram-positive isolates were defined as AHL-producers. All Gram-negative isolates, formerly defined as non-AHL producers by both biosensors and HPLC methods, were identified as AHL-producers. DFD signal was only detected from Gram-negative Klebsiella pneumonia, Enterobacter cloacae and Klebsiella oxytoca isolates. The results demonstrated that the QS-system is a complex system and biosensor microorganism may not be the best method for QS-signal identification. The results also provided new insights in defining the profile and chemical composition of QS-signals importance for interrupting the chemical communication completely to reduce biofilm formation and prevent resistance gain of microorganisms.

Biocide Syntheses Bee Venom-Conjugated ZnO@αFe2O3 Nanoflowers as an Advanced Platform Targeting Multidrug-Resistant Fecal Coliform Bacteria Biofilm Isolated from Treated Wastewater

This study targeted developing a novel Zinc oxide with alpha hematite nanoflowers (NFs)-loaded bee venom (Bv) (Bv-ZnO@αFe2O3 NFs) as a bio-natural product from bees to combine both the advantages of combination magnetic properties and the antimicrobial and anti-biofilm properties on isolated coliform bacteria from the effluent of wastewater treatment plants. About 24 isolates of treated wastewater isolates were multidrug resistant (MDR). The phylogenetic grouping of Escherichia coli (E. coli) and Klebsiella pneumonia (K. pneumonia) showed that the largest group was Group A, followed by Group B2 and Group B1. Fourier transform infrared (FTIR), The X-ray diffraction (XRD), and scanning electron microscopy-energy dispersive X-ray analysis (SEM− EDX) validated the coating operation’s contact with Bv onto ZnO@αFe2O3 NFs. According to high-resolution transmission electron microscopy (TEM) and selected area electron diffraction (SAED), pattern analyses for prepared nanoformulations exhibited a spherical shape of αFe2O3 (~9–15 nm), and floral needle shapes with uniform distribution of size with aggregation of ZnOαFe2O3 and Bv-ZnO@αFe2O3 NFs around (~100–200 nm). The toxicity of Bv-ZnO@αFe2O3 NFs was comparable up to 125 µg mL−1, when it reached 64.79% (IC50, 107.18 µg mL−1). The antibacterial activity showed different zones of inhibition against different isolates. The biofilm inhibitory activity of NPs and NFs showed a highly significant reduction (p < 0.001) in treated biofilms with ZnO@αFe2O3 and Bv-ZnO@αFe2O3. In essence, ZnO@αFe2O3 and Bv-ZnO@αFe2O3 NFs are promising antimicrobials for inhibiting the growth and biofilm of MDR E. coli and K. pneumonia isolates, thereby, biocontrol of wastewater.

Effect of Berberine as efflux pump inhibitor in multidrug-resistant Klebsiella pneumoniae isolated from urinary tract infections

The urinary tract infections with K. pneumoniae have increased over many years. The emergence of antibiotic resistance among these bacteria presents a challenging problem for the clinician regarding the management and treatment of infections. The multidrug resistance in K. pneumoniae is due to several mechanisms, one of which is the role of efflux pumps. The current study investigated the role of Efflux Pump Inhibitors Phenylalanine-Arginine β-Naphthylamide (PAβN) and Berberine as antibacterial agents with multidrug-resistant K. pneumonia isolates from urinary tract infections. The collection of study samples took place between December 2021 and completed at the end of April 2022; it included 260 urine samples collected from outpatients and inpatients suffering from urinary tract infections during this period, from both genders with ages ranging from 15 to 72 years in five hospitals in Baghdad. The results of selective media, biochemical tests, and the VITEK2 system identified 76 isolates (65.5%) as K. pneumoniae from all collected bacterial cultures. The results of the antimicrobial susceptibility test using the disc diffusion method for the isolates under study showed that K. pneumoniae clinical isolates were moderately resistant to most antibiotics tested. Most K. pneumoniae isolates were highly resistant to Amoxicillin (96.1%) and Trimethoprim (80.3%). Also, there was apparent resistance to Gentamicin and Amikacin, while the lowest percentage of resistance was for Meropenem (55.1%) and Ciprofloxacin (53.9%). The susceptibility of the strains to Ciprofloxacin was highly increased in the presence of the efflux pump inhibitor (PAβN). The PAβN reduced the minimum inhibitory concentrations (MICs) by 4- to 64-fold. The results of minimum inhibitory concentrations (MICs) of Berberine against ten K. pneumoniae isolates with multidrug resistance revealed that the range of MICs of Berberine was (3.9-500 µg/ml) and it was obvious that there is a significant effect of Berberine on the growth of K. pneumoniae at deficient concentrations. This study concluded that using Berberine as an efflux pump inhibitor and antimicrobial agent may become a new generation of urgently needed antimicrobials that can overcome bacterial resistance mechanisms. Keywords: UTI infections, MDR, Berberine, Klebsiella pneumoniae

Design, synthesis, and unraveling the antibacterial and antibiofilm potential of 2-azidobenzothiazoles: insights from a comprehensive in vitro study.

The present study reports the synthesis of 2-azidobenzothiazoles from substituted 2-aminobenzothiazoles using sodium nitrite and sodium azide under mild conditions. All the synthesized compounds were examined for their antibacterial activity against Gram (+) bacteria, Staphylococcus aureus (ATCC 25923), Enterococcus faecalis (ATCC 51299), Bacillus cereus (ATCC 10876) and Gram (-) bacteria, Escherichia coli (ATCC 10536), Pseudomonas aeruginosa (ATCC 10145), Klebsiella pneumonia (ATCC BAA-2146)and clinical isolates of Gram (+) Methicillin Resistant S. aureus (MRSA) and Multi Drug Resistant E. coli. The Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) values by broth dilution method revealed that compound 2d exhibited significant antibacterial potential against E. faecalis and S. aureus with MIC of 8μg/mL, while other synthesized compounds had only moderate effects against all the tested species. The compound significantly inhibited the biofilm formation of the bacterial strains below its MIC. The selective cytotoxicity of Compound 2d towards bacterial cells was evidenced on extended exposure of Human Embryonic Kidney-293 cell line to higher concentrations of the compound. Hence, the present study confirmed that compound 2d can be a potential drug candidate for future development as an antibacterial drug.

Evaluation of the Inhibitory Activity of Syzygium aromaticum Extract -Chitosan Nanoparticles Against Biofilm Formation of Klebsiella pneumonia

The increasing resistance of Klebsiella pneumoniae to antibiotics has led to difficulties in treating infections due to its virulence factors. As one of its major pathogenic factors, this opportunistic pathogen may develop a thick biofilm coating, allowing the bacteria to attach to living or nonliving surfaces and promote drug resistance. Searching for therapeutic alternatives from a plant source that was safe and effective in treating this multi-drug-resistant bacteria was necessary. In this concept, Syzygium aromaticum extract (SAE) is used to combat K. pneumonia. The extract was confirmed by GC-MS and loaded onto chitosan nanoparticles (SACSNPs). The SACSNPs were prepared by the ionic gelation method with tripolyphosphate (TPP). And then characterized using UVvis, FTIR, AFM, SEM, and XRD techniques. The K. pneumonia isolates were obtained and identified using the VITEK-2 system. The MIC of SAE and SACSNPs were confirmed using a 96-well resazurin-aided microdilution method, which was 6.25 μg/ml for SACSNPs and 75.5 μg/ml for SAE. The inhibitory activity using sub-MIC of analytical substances was determined by measuring the optical density using a microplate reader with a 96-well plate and 0.1% crystal violet dye. The results show that the S. aromaticum extract loaded with chitosan nanoparticles has higher inhibitory activity against the biofilm formation of K. pneumonia than the S. aromaticum extract. Keywords: Chitosan nanoparticles, S. aromaticum, K. pneumonia, Biofilm, GCMS, resazurin, XRD.