Isolates Of Gram-negative Bacteria Research Articles

Occurrence and Associated Risk Factors of Metallo-B-Lactamase Producing Agents of Infection among Patients in Uyo, Southern Nigeria

Background: Carbapen­emase enzymes such as metallo-β-lactamase (MβL) belong to Amber class B type of β- lactamase that cause serious clinical infections mostly feared due to the associated antibiotic resistance mechanisms resulting from their ability to hydrolyse virtually all β-lactam agents, including the carbapenems. Aim: To investigate the occurrence as well as determine the associated risk factors of metallo- β -lactamase producing bacterial agents of infection among in Uyo, Southern Nigeria. Methods: A clean-catch, mid-stream urine and wound samples were collected from consented patients and cultured according to standard procedure. The isolated pathogens were subjected to antibiotic susceptibility testing using the Kirby Bauer method. All Imipenem and Ertapenem resistant isolates were used for of MβL detection, using Total Metallo-β-Lactamase confirmation Kit. Results: Of the 157 Gram-negative bacteria isolates from 312 clinical samples of urine and wound, metallobeta-lactamase (MβL) was detected in 12 (7.6%). The highest MβL producing Gram-negative bacteria isolate from urine was Escherichia coli. However, Pseudomonas aeruginosa was the major producer from wound the samples. Overall, the antibiotic sensitivity profile revealed that the isolates were mostly sensitive to imipenem 123(91.0%) and highly resistant to the commonly used antibiotics in the study area. The associated risk factors of note were the participants residence, antibiotic use and duration of wound infection and they were found to be statistically significant (P = <0.05). Conclusion: The prevalence of metallo-ß-lactamase producing bacterial agents in Uyo, Nigeria was found to be 7.6%. In addition, while E. coli and P. aeruginosa were the major MβL producers, the participant’s place of residence, antibiotics use and wound duration were found to be associated with their occurrence.

Prevalence and antibiogram of non-urinary isolates among patients attending a tertiary care hospital

Introduction and Aim: Gram-negative bacterial infections have emerged as a global health problem in clinical care. They account for the majority of community and hospital acquired infections which are resistant to various antimicrobials. The present study aimed to investigate the prevalence and antibiotic susceptibility pattern of Gram-negative bacteria (GNB) isolated from various non-urinary samples at a tertiary care hospital in Haryana. Materials and Methods: A total of 228 isolates were obtained from various clinical samples from January 2018 to July 2018 and were identified by standard bacteriological techniques. Antibiotic susceptibility testing was done using the disc diffusion technique by the Kirby Bauer method. Results: Adult patients showed higher isolation of GNB (31%) as compared to paediatric patients (16.3%). The most common GNB isolated from blood was Pseudomonas spp.(62%) and Klebsiella spp.(66.7%) from respiratory samples isolated in our study showed resistance to common antibiotics like gentamicin and ceftriaxone. Polymyxin B and Tigecycline were found to be sensitive. Conclusion: Strict compliance with antimicrobial stewardship policy should be followed to reduce the menace of antibiotic resistance.

The association between biofilm formation ability and antibiotic resistance phenotype in clinical isolates of gram-negative bacteria: a cross-sectional study

Backgrounds: Antimicrobial-resistant pathogens that form biofilms cause treatment failure, increased mortality, and morbidity. Gram-negative bacteria (GNB) infections have become an emerging issue in antimicrobial resistance. This study analyzes the association between GNB biofilm formation ability and the antibiotic resistance phenotype in GNB clinical isolates. Methods: From June to August 2022, this cross-sectional study was conducted at Dr. Soetomo General Academic Hospital. Isolates derived from clinical specimens, including blood, urine, and respiratory tract specimens, were identified for antibiotic resistance phenotypes along with a quantitative biofilm formation assay. The antibiotic resistance phenotype was studied in relation to the GNB isolates' ability for biofilm formation. Results: Of the 271 isolates studied, 95 (35.1%) were non-MDR, 143 (52.8%) were MDR, and 33 (12.2%) were XDR. On the biofilm formation assay, 34 (12.5%) were non-biofilm producers, 237 (87.5%) were biofilm producers, consisting of 101 (37.3%) weak, 131 (48.3%) moderate, and 5 (1.8%) strong biofilm producers. Biofilm formation and antibiotic resistance in Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii were not significantly associated (p > 0.05). However, Escherichia coli isolates resistant to the antibiotics amikacin, imipenem, meropenem, and amoxicillin-clavulanate were found to have a higher predisposition to form biofilms. Klebsiella pneumoniae isolates resistant to gentamycin were also positively associated with biofilm formation. Conclusions: Gram-negative bacteria's ability to form biofilms is not associated with their antibiotic resistance phenotype; therefore, routine biofilm monitoring is critical for enhancing the quality of treatment strategy for biofilm-associated infection.

Isolation and Identification of Gram-Positive and Gram-Negative Bacteria from Infertility Patients in Tikrit City

The objectives of the current study were conducting a statistical study to find out the number of men with primary and secondary infertility, immune infertility, and unexplained infertility in the city of Tikrit. Isolation and identification of Gram-positive and Gram-negative aerobic bacteria, and the study of some patterns of sensitivity to antibiotics of the isolated bacteria. The results showed that the percentage of primary infertility was the highest among infertility diseases, as the percentage reached 57%, while the lowest percentage was for immunological infertility, as it amounted to 7%. While the percentage of secondary and unexplained infertility was 15% and 31%, respectively. About the bacteriological study, the results showed two types of isolates, single bacterial isolates (52%), mixed bacterial isolates (38%). Percentages of isolates from E. coli and Klebsiella spp. and Staphylococcus spp. and Streptococcus spp. were 100%, 12%, 18%, and 10%, respectively. E. coli showed total (100%) resistance to amoxicillin/clavulanic acid and ampicillin. 50% resistance to Nalidixic. Otherwise, Escherichia coli showed resistance of 40%, 20%, 60%, 20% and 50% to Imipenem, Amikacin, Gentamicin, Azithromycin and Cefepime respectively. Klebsiella showed total (100%) resistance to amoxicillin/clavulanic acid and ampicillin. 40% resistance to Nalidixic. Otherwise, Klebsiella spp. showed resistance of 20%, 10%, 10%, 40% and 20% to imipenem, amikacin, gentamicin, azithromycin and cefepime respectively. Staphylococcus aureus showed complete resistance to penicillin, oxacillin and cefoxitin. 10%, 0%, 30%, 30%, 0%, 90% and 50% are resistant to gentamicin, nitrofurantoin, erythromycin, azithromycin, rifampicin, trimethoprim and ciprofloxacin respectively. Streptococcus spp. completely resistant to penicillin, oxacillin and cefoxitin. 30%, 0%, 20%, 10%, 0%, 70% and 30% are resistant to gentamicin, nitrofurantoin, erythromycin, azithromycin, rifampicin, trimethoprim and ciprofloxacin respectively.

A Clinico-Microbiological Study of Blood Stream Infections in a Tertiary Referral Hospital: Colistin Resistance & Challenges

Carbapenems, frequently used for the treatment of infections caused by Multidrug-resistant (MDR) Gram-negative bacteria (GNB) are being reported with increased resistance rate. Colistin with other antibiotics has emerged as a saviour but inappropriate reporting of colistin susceptibility is a serious clinical concern. To detect the antimicrobial resistance of GNB isolates obtained from blood samples, further, colistin Minimum Inhibitory Concentration (MIC) testing of carbapenem-resistant isolates was done by the Broth Micro-dilution Method (BMD). This prospective study was done in the Department of Microbiology from January 2020 to December 2020. The GNB isolated from blood samples were identified & antimicrobial-susceptibility testing was performed by the Vitek 2 system. Colistin MIC of carbapenem-resistant isolates was done by the BMD method. The data were statistically analysed using SPSS 21. Growth was obtained in 5% of blood samples and 546 (75.8%) of these were GNB including predominantly E.coli, Klebsiella spp & Acinetobacter spp. Carbapenem-resistant infections 246 (48.5%) showed significant association with ICU admission, resistance to other classes of antibiotics & mortality. Among the carbapenem-resistant isolates, only seven (2.9%) were found resistant to colistin by the BMD test. Most of these were Klebsiella spp. (71.4%) & obtained predominantly from ICU patients (85.7%). All the carbapenem-resistant isolates were found intermediate sensitive to colistin by the VITEK-2 system. The isolates of GNB were characterized as MDR 323 (59.2%), XDR 164 (30%) and PDR 2 (0.4%). Use of, colistin, should be guided by BMD, the reference method for MIC testing to avoid erroneous reporting of colistin resistance.

PREVALENCE AND PHENOTYPIC CHARACTERIZATION OF MULTI-DRUG-RESISTANT ISOLATES CAUSING WOUND INFECTIONS IN A TERTIARY CARE CENTRE

Objective: Wound infections lead to 70-80% mortality among post-surgeries and one-third of nosocomial infections. The prolonged hospitalization due to diagnostic tests, complete antibiotic course, and clearing of wound infection all together increase the healthcare cost.Methods: The present study was a cross-sectional study carried out in the Department of Microbiology, Central laboratory, and teaching hospital from May 2022 to October 2022. All consecutive, non-duplicate gram-positive and gram-negative bacteria isolates were collected from pus and wound swabs from outpatients and hospitalized patients during the study period.Results: A total of 260 isolates from various wound swabs and pus samples were collected from March 2022 to August 2022. Species-wise distribution of organisms along with antibiotic susceptibility testing shows that 15 out of 63 (24%) Escherichia coli, 12 out of 38 (31.5%) Klebsiella pneumoniae, 06 out of 29 (20.6%) P. aeruginosa, 06 out of 09 (40%) Acinetobacter baumannii, 05 out of 08 (62%) Klebsiella oxytoca, 04 out of 12 (33%) Citrobacter freundii, 01 out of 07 (14.3%) Enterobacter aerogenes were multi-drug-resistant (MDR). Previously few studies mentioned S. aureus was predominant, followed by P. aeruginosa in polymicrobial wound infections. Our study found that around 2-3% of cultures showed two organisms. The antibiotics like amikacin and imipenem worked well against all gram-negative organisms up to 72%, and 85%, respectively. Similar findings of organisms in other studies showed sensitivity to amikacin and imipenem up to 77% and 100%; 70% and 83%, respectively.Conclusion: The organisms causing wound infections and the empirical therapy and switch to correct antibiotics as soon as possible to avoid misuse of antimicrobials and prevent the spread of drug-resistant strains among the community and hospital setup.

Evaluation of Early Reading of Broth Microdilution Technique for Polymyxin B.

Delay in the results of standard phenotypic susceptibility tests is the main obstacle to adequate antibiotic treatment. For this reason, the European Committee for Antimicrobial Susceptibility Testing has proposed the Rapid Antimicrobial Susceptibility Testing for the disk diffusion method directly from blood culture. However, to date, there are no studies evaluating early readings of polymyxin B broth microdilution (BMD), the only standardized methodology for assessing susceptibility to polymyxins. This study aimed to evaluate modifications in the BMD technique for polymyxin B using fewer antibiotic dilutions and reading after an incubation time of 8-9 hr (early reading) in comparison to 16-20 hr of incubation (standard reading) for isolates of Enterobacterales, Acinetobacter baumannii complex, and Pseudomonas aeruginosa. A total of 192 isolates of gram-negative bacteria were evaluated and the minimum inhibitory concentrations were read after early and standard incubations. The early reading presented 93.2% of essential agreement and 97.9% of categorical agreement with the standard reading of BMD. Only three isolates (2.2%) presented major errors and only one (1.7%) presented a very major error. These results indicate a high agreement between the early and the standard reading times of BMD of polymyxin B.

Prevalence and Phenotypic and Molecular Characterization of Carbapenemase-Producing Gram-Negative Bacteria in Gabon.

Data collection and monitoring of carbapenemase-producing (CP) Gram-negative bacteria (GNB) are often limited. This study determined CP-GNB prevalence in Gabon and the genetic origins of the resistance genes. From January 2016 to March 2018, 869 clinically significant GNB isolates from inpatients and outpatients, and 19 fecal samples (inpatients) were analyzed in the main hospitals of Gabon. Fecal samples were screened using ChromID® CARBA SMART selective chromogenic medium biplates. Species were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Antibiotic susceptibility was tested using the disk diffusion method on Müller-Hinton agar, and resistance genes were assessed by multiplex polymerase chain reaction and sequencing. Overall, 1.61% of clinical isolates (14 of 869) and 5.26% of fecal samples (1 of 19) were CP-GNB. The CP-GNB rate was higher among inpatients (2.98%) than outpatients (0.33%), in intensive care units (28.57%, 4 of 14), and in urine samples (35.71%, 5 of 14). The most common CP-GNB were Klebsiella pneumoniae (53.33%) and Acinetobacter baumannii (26.67%). blaOXA-48 was the predominant carbapenemase-encoding gene (40%), followed by blaNDM-5 (33.33%). The A. baumannii multilocus sequence types ST2 and ST78, Enterobacter cloacae ST78, Escherichia coli ST2, and K. pneumonia ST48 and ST147 were found. These data indicate that CP bacteria are present in clinical and carriage samples. Preventive measures are needed to avoid the spread of resistance genes.

Prevalence of Carbapenem Non-susceptible Gram-Negative Bacteria at Tertiary Care Hospitals in Saudi Arabia.

Background Antibiotics significantly increased life expectancy and decreased mortality rates due to infections. However, this trend is starting to fade with the rise of multidrug-resistant organisms (MDR); these strains are becoming a global burden on healthcare and the economy. The dramatic increase and spread of carbapenem-resistant gram-negative bacteria (CRGNB) has become a serious global public health concern. In this retrospective cross-sectional study, we aimed to estimate the rates of gram-negative bacteremia in five tertiary care hospitals in different geographical locations in Saudi Arabia for five years. Methods A retrospective cross-sectional study was conducted in five tertiary care hospitals in Saudi Arabia among patients with bacteremia due to CRGNB. Electronic medical records were used to retrieve data regarding patient demographics and antimicrobial susceptibility testing (AST) over five years between January 2016 and December 2020. Patients with positive blood cultures for carbapenem-resistant Escherichia (E.) coli, Klebsiella (K.) pneumonia, Pseudomonas (P.) aeruginosa, andAcinetobacter (A.) baumannii comprise the final study population. Results This retrospective multicentric study was conducted between 2016 and 2020 in five tertiary care hospitals across five cities in Saudi Arabia. E. coli (n=2190, 38.03%), K. pneumoniae (n=2154, 37.41%), P. aeruginosa (n = 918, 15.94%), and A. baumannii (n=496, 8.61%) constitutethe 5758 gram-negative bacteria isolates. E. coli was the most frequently identified species in Riyadh, AlAhsa, Dammam, and Madinah (40%, 46.50%, 61.67%, and 43.66%, respectively), with a p-value of (p<0.001), except in Jeddah, where K. pneumoniae was the most prevalent (42%). The mean age of patients across Riyadh, AlAhsa, Dammam, and Madinah was 62.2 years (± 4.24). In contrast to Jeddah, where the majority of isolates (702; 41.8%)belonged to the adult age group. Most isolates were from male patients (3045; 52.9%), compared to 2713 (47.1%) from female patients. K. pneumoniae 1226 (40.3%) was the most prevalent isolate among male patients while E. coli(1135; 41.8%) was the most prevalent isolate among female patients. Conclusion Our study showed that the prevalence of carbapenem non-susceptible Gram-negative bacteria is relatively high, which therefore makes them very challenging to treat. The results show an urgent need for improved antibiotic stewardship strategies, including better surveillance and more effective infection control measures to reduce this issue. Further research into the molecular epidemiology and risk factors associated with these infections is necessary to guide public health policymakers in developing interventions to help control the spread of carbapenem-resistant Gram-negative bacteria.

Development of a novel loop-mediated isothermal amplification assay for ß-lactamase gene identification using clinical isolates of Gram-negative bacteria.

Rapid evaluation of antimicrobial susceptibility is important in the treatment of nosocomial infections by Gram-negative bacteria, which increasingly carry carbapenemases and metallo-β-lactamases. We developed loop-mediated isothermal amplification (LAMP)-based assays for four β-lactamase genes (bla KPC, bla NDM-1, bla IMP-1 group, and bla VIM). The assays were evaluated using eight reference bacterial strains (Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter bereziniae) harboring six β-lactamase genes. A total of 55 Gram-negative bacterial strains, including 47 clinical P. aeruginosa isolates, fully characterized by next-generation sequencing (NGS), were used to evaluate the LAMP assays. The results were compared to those of conventional PCR. The LAMP assays were able to detect as few as 10 to 100 copies of a gene, compared to 10 to 104 copies for conventional PCR. The LAMP assay detected four β-lactamase genes with a sensitivity similar to that using purified DNA as the template in DNA-spiked urine, sputum, and blood specimens. By contrast, the sensitivity of PCR was 1- to 100-fold lower with DNA-spiked clinical specimens. Therefore, the LAMP assays were proved to be an appropriate tool for the detection of four β-lactamases.

Retracted: In Vitro Activity of Various Sulbactam Compounds and Piperacillin/Tazobactam against Clinical Isolates of Different Gram-Negative Bacteria.

[This retracts the article DOI: 10.1155/2021/1175379.].

Isolation, identification and antibiotic susceptibility of gram-negative anaerobic bacteria in oral cavity

Gram-negative bacteria include more than 20 genera. The most commonly isolated genera are: Bacteriodes spp., Porphiromonas spp., Fusobacterium spp. and Prevotella spp. The following genera were isolated somewhat less frequently: Tanerella spp., Leptotricha spp., Veilonella spp.,Wollinela spp., Selenomonas spp. and Treponema spp. Anaerobic bacteria have an anaerobic type of metabolism and therefore their incubation is significantly longer and more demanding than aerobic bacteria. The genera Prevotella spp., Porphyromonas spp. and Fusobacterium spp. are part of the resident flora of the oral cavity and in unfavorable conditions cause periodontal diseases, and sometimes dentogenic infections and systemic diseases, such as Alzheimer?s disease, cardiovascular diseases, metabolic diseases and inflammatory bowel diseases. The three most important steps for the successful diagnosis of anaerobic bacteria are: proper sampling with avoiding sample contamination, rapid transport of samples to the microbiological laboratory and proper handling of samples. A combination of beta-lactam with the addition of beta-lactamase inhibitors, metronidazole, clindamycin and moxifloxacin is used for treatment for infections caused by anaerobic Gram-negative bacteria. It is important to note that antibiotics should be used only with a clear indication and to choose the right antibiotic in the optimal dose. The aim of this review is to point out the role of Gram-negative anaerobic bacteria in periodontal diseases, and its isolation, identification and antibiotic susceptibility.

Detection and prevalence of extended spectrum β-lactamases in gram-negative bacteria at our health-care center

Background: Emergence of extended spectrum β-lactamase (ESBL) producing strains of Gram-negative bacteria can lead to serious infections frequently complicates the clinical and treatment outcome. Aims and Objectives: The purpose of this study was to know the prevalence of ESBLs and to know the most common Gram-negative bacteria, which produce ESBLs at our health-care facility. Materials and Methods: This study comprised all of the isolates of Gram-negative bacteria that were acquired from various clinical samples. For the purpose of the investigation, a sample of an isolate that showed resistance to two or more third-generation cephalosporins was taken. ESBL detection and antibiotic sensitivity tests were carried out using conventional microbiological techniques. Results: We isolated a total of 284 Gram-negative bacteria and 54 (19%) were identified as ESBL producers. Out 54 ESBL producers, 18 (33%) isolates were Escherichia coli, 11 (20%) were Pseudomonas aeruginosa, 12 (22%) were Klebsiellae, 6 (11%) were Enterobacter, 2 (4%) were Citrobacter, 4 (8%) were Acinetobacter, and 1 (2%) were Serratia spp. Conclusion: Clinical decision-makers can make the best antibiotic treatment by regularly monitoring multi-drug resistant bacteria like ESBL producers. This also helps to improve infection control procedures. In addition, we must maintain reserve medications like carbapenems on hand for judicial use.

Biofilm Formation and its Association with Gram Negative Sepsis Pathogenicity

Background and objectives: Biofilm formation is an important virulence factor that protects an organism from antimicrobial agents as well as host immune effectors, thus allowing organisms to invade, survive, and cause persistent-reoccurring infection in host cells. The aim of this study was to investigate the ability of sepsis-causing gram-negative bacteria to form biofilms, evaluate the association between antibiotic resistance pattern and biofilm formation, determine the role and influence of biofilm formation on pathogenicity and clinical outcome of sepsis. Methods: A prospective study conducted from October 2020 to August 2021, non-replicated gram-negative bacteria isolates were recovered from blood samples of patients with suspected bacteremia, sepsis, and sepsis shock and identified using biochemical procedures. Antimicrobial susceptibility patterns of GNB isolates were determined using the Kirby-Bauer disc diffusion method and interpreted using CLSI guidelines. The ability of GNB isolates to form biofilm was assessed using Congo red agar and the tissue culture plate method. Results: Of the 160 Gram-negative bacteria tested, biofilm formation was seen in 73 (45.63%) isolates. Isolates are Klebsiella pneumoniae (39.73%), Acinetobacter spp. (34.25%), Escherichia coli (23.29%), Pseudomonas aeruginosa (1.37%), and other non-fermenters (1.37%). Isolates were highly resistant to cephalosporins, fluoroquinolones, and the penicillin group of antibiotics. No statistical relationship was found between resistance pattern, clinical outcome, and biofilm formation. Conclusion: In the current study, we found that 45.63% of gram-negative bacteria causing sepsis were biofilm producers. Klebsiella pneumonia isolates exhibited the highest levels of biofilm formation and antimicrobial resistance. Based on the strength of biofilm formation, most isolates were weak biofilm producers, and there was no statistical correlation between the formation of biofilms and antimicrobial resistance, indicating that the formation of biofilms was not a determining factor for resistance.

Prevalence of multidrug-resistant Gram-negative bacteria from blood cultures and rapid detection of beta-lactamase-encoding genes by multiplex PCR assay.

This study aimed to determine the prevalence of multidrug-resistant Gram-negative bacteria (GNB) from blood cultures in a tertiary-care hospital and the multiplex PCR assay's ability to detect resistance genes. A total of 388 GNB isolates obtained from hospitalized patients between November 2019 and November 2021 were included in the study. Antimicrobial susceptibility testing was done by VITEK 2 system and broth microdilution method. Beta-lactamase-encoding genes were detected by multiplex PCR assays, BioFire-Blood Culture Identification 2 (BCID2) panel (bioMérieux, France). Extended-spectrum beta-lactamases (ESBLs) were detected phenotypically with VITEK AST-GN71 card (bioMérieux, France). The isolates of GNB were classified into multidrug-resistant, extensively-drug-resistant, and pandrug-resistant categories, and their prevalence and distribution in different wards, including coronavirus diseases 2019 (COVID-19) intensive care units (ICU), were calculated. Results revealed that all isolates of Acinetobacter baumannii and Pseudomonas aeruginosa were multidrug-resistant as well as 91.6% of Enterobacter cloacae, 80.6% of Proteus mirabilis, and 76.1% of Klebsiella pneumoniae, respectively. In fermentative bacteria, blaOXA-48-like (58.1%), blaNDM (16.1%), blaKPC (9.7%) and blaVIM (6.5%) genes were detected. More than half of Enterobacter cloacae (58.3%) and Klebsiella pneumoniae (53.7%) produced ESBLs. Among non-fermenters, the blaNDM gene was carried by 55% of Pseudomonas aeruginosa and 19.5% of Acinetobacter baumannii. In the COVID-19 ICU, Acinetobacter baumannii was the most common isolate (86.1%). This study revealed high proportions of multidrug-resistant blood isolates and various underlying resistance genes in Gram-negative strains. The BCID2 panel seems to be helpful for the detection of the most prevalent resistance genes of fermentative bacteria.

Third-generation cephalosporin resistant gram-negative bacteraemia in patients with haematological malignancy; an 11-year multi-centre retrospective study

ObjectivesAmong patients with haematological malignancy, bacteraemia is a common complication during chemotherapy-induced neutropenia. Resistance of gram-negative bacteria (GNB) to third-generation cephalosporins (3GC) is increasing. In order to explore the value of using surveillance cultures to guide empirical treatment e.g. choosing between carbapenem versus ceftazidime- we aimed to assess the distribution of pathogens causing bacteraemia in patients with haematological malignancy, and the proportion of 3GC-resistant GNB (3GC-R GNB) bacteraemia that was preceded by 3GC-R GNB colonization.MethodsUsing 11 years of data (2008–2018) from the Dutch national antimicrobial resistance surveillance system, we assessed the prevalence of 3GC-R GNB in episodes of bacteraemia, and the proportion of 3GC-R GNB bacteraemia that was preceded by 3GC-R GNB colonization. Colonization was defined as availability of any GNB surveillance isolate in the year before, independent of the causative micro-organism (time-paired isolates).ResultsWe included 3887 patients, representing 4142 episodes of bacteraemia. GNB were identified in 715/4142 (17.3%), of which 221 (30.9%) were 3GC-R GNB. In 139 of these 221 patients a time-paired surveillance culture was available. In 76.2% (106/139) of patients these surveillance cultures already showed 3GC-R GNB isolates in the year prior to the culture date of the 3GC-R GNB positive blood isolate.ConclusionsThis multi-centre study shows that in patients with haematological malignancy, the majority of 3GC-R GNB bacteraemia is preceded by 3GC-R GNB colonization. Prospective clinical studies are needed to assess the safety and benefits of the use of surveillance-cultures to guide empirical therapy to restrict the empirical use of carbapenems in this population.

The Antibacterial Activity of Kaempferol Combined with Colistin against Colistin-Resistant Gram-Negative Bacteria.

Social hygiene is seriously threatened by the rise in colistin (COL) resistance against Gram-negative bacteria (GNB). With resistance to last-line antibiotics such as COL becoming more common, it is imperative to identify alternative treatment options. In our work, we sought to determine if COL plus kaempferol (KP) present synergistic effects on the antibacterial and antibiofilm activities against colistin-resistant (Col-R) GNB in vivo and in vitro. Twenty-four Col-R GNB were collected as the experimental strains. The synergistic activity of COL and KP was evaluated by checkerboard method, time-killing assays, and the Galleria mellonella experiment. The antibiofilm effectiveness of the COL/KP combination against Col-R GNB was assessed using biofilm inhibition and eradication assays and scanning electron microscopy (SEM). Cytotoxicity tests were performed to detect the toxicity of KP monotherapy or combination therapy. There is synergistic antibacterial activity of COL and KP combination in vitro. KP combined with COL could inhibit the formation of bacterial biofilms. The amalgamation of COL and KP considerably reduced the amount of bacteria in the biofilm, according to the SEM findings. The COL/KP combination improved the survivorship of infected larvae in the G. mellonella in vivo infection model. In addition, the combination of KP and COL showed no cytotoxicity at synergistic combined concentrations according to cytotoxicity assays. This represents the first account of the antibacterial and antibiofilm activities of KP in combination with COL against Col-R GNB. Therefore, our results may provide an effective alternative route to combat Col-R GNB infections. IMPORTANCE COL is one of the few antibiotics effective against clinical isolates of GNB. However, in recent years, GNB resistance to colistin has been increasing. As a result, the combined application of colistin in conjunction with nonantibacterial medications has garnered considerable interest. In this work, the KP/COL combination showed effective antibacterial and antibiofilm activities in vitro and in vivo. The synergistic effect of combined application may be attributed to membrane permeability. Due to the low cytotoxicity of the combined concentration, the combination exhibits a promising future for use in clinical anti-infection treatments. This finding might broaden the potential applications for COL.

Microfluidic Acoustophoresis for Flowthrough Separation of Gram-Negative Bacteria using Aptamer Affinity Beads.

This article describes the fabrication and operation of microfluidic acoustophoretic chips using a microfluidic acoustophoresis technique and aptamer-modified microbeads that can be used for the fast, efficient isolation of Gram-negative bacteria from a medium. This method enhances the separation efficiency using a mix of long, square microchannels. In this system, the sample and buffer are injected into the inlet port through a flow controller. For bead centering and sample separation, AC power is applied to the piezoelectric transducer via a function generator with a power amplifier to generate acoustic radiation force in the microchannel. There is a bifurcated channel at both the inlet and outlet, enabling simultaneous separation, purification, and concentration. The device has a recovery rate of >98% and purity of 97.8% up to a 10x dose concentration. This study has demonstrated a recovery rate and purity higher than the existing methods for separating bacteria, suggesting that the device can separate bacteria efficiently.

Molecular Screening of Clinical Multidrug-Resistant Gram-Negative Bacteria Shows Endemicity of Carbapenemases, Coexistence of Multiple Carbapenemases, and Rarity of mcr in South Africa.

Background: Extensive use of carbapenems to treat multidrug-resistant (MDR) Gram-negative bacteria (GNB) facilitates the wide dissemination of carbapenemase-producing carbapenem-resistant GNB. Colistin was reintroduced into clinical settings to manage these GNB infections. However, there is currently an increase in the dissemination of mobile colistin resistance (mcr)-producing colistin-resistant GNB isolates in clinical settings. The epidemiology of carbapenemases and mcr in Pretoria was evaluated. Methods: Clinical MDR GNB were collected and screened for carbapenemases and mcr using polymerase chain reaction (PCR); their antibiotic susceptibility profiles were elucidated using the Vitek® 2 automated system (Biomerieux, France) and microbroth dilution (for colistin). Results and Discussion: A total of 306 isolates were collected; a majority of these were Klebsiella pneumoniae (n = 208) and were collected from males (n = 158). The isolates were retrieved from a variety of infection sites, including urine, blood cultures, and rectal swabs. The Vitek 2 system found that these isolates were largely resistant to β-lactams, where 217 (70.9%) had reduced susceptibility to at least one carbapenem (ertapenem, meropenem, or imipenem), and 81 isolates (26.5%) were resistant to colistin. PCR screening identified 201 (65.7%) isolates harboring carbapenemase genes consisting of blaOXA-48 (170, 84.2%), blaNDM (31, 15.4%), blaIMP (5, 2%), blaKPC (4, 1%), and blaVIM (5, 2%). Furthermore, 14 blaOXA-48-producing isolates were coharboring blaVIM (2), blaNDM (9), blaKPC (1), and blaIMP (2) genes. Only one isolate harbored the mobile colistin resistance (mcr)-1 gene, and this is the first report of an mcr-1-producing Acinetobacter baumannii isolate in South Africa. Conclusion: There is high endemicity of carbapenemase genes and a low prevalence of mcr genes in GNB, particularly in K. pneumoniae, in health care facilities in Pretoria and surrounding regions of South Africa. Significance: Health care facilities in Pretoria are becoming breeding grounds for MDR infections that threaten public health. Careful use of carbapenems and other antibiotics is necessary to prevent further escalation and outbreak of these MDR strains that can claim several lives.

Isolation and Genetic Characterization of Gram-negative Bacteria from Different Fresh Retail Vegetables in Nigeria

Aim: The proportion of food borne disease outbreaks as a result of contaminated products has increased over the years. In this study, the genetic characteristics of antibiotic resistant Gram-negative bacteria from different fresh retail vegetables in Okada, Edo state Nigeria was investigated.&#x0D; Place and Duration of Study: In April-May 2021, the study was carried out in the Department of Pharmaceutical Microbiology, Igbinedion University Okada Edo state Nigeria.&#x0D; Methodology: One hundred and eight isolates were isolated from sixteen different retail leafy and salad vegetable samples. Recovered isolates from samples were identified using standard microbiological techniques. Species identification for ten randomly selected isolates was performed by Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry and ribosomal multilocus sequence typing (rMLST). Antimicrobial susceptibility testing was performed using the Kirby-Bauer method for 15 antibiotics. Isolates were characterized by whole genome sequencing (WGS).&#x0D; Results: Species identification using MALDI-TOF-MS and ribosomal MLST assigned the 10 randomly selected isolates to four different species. Identified isolates include Proteus mirabilis, Proteus vulgaris, Acinetobacter baumanii and Klebsiella quasipneumoniae. Out of the 10 randomly selected isolates, 60% (6/10) were antibiotic resistant in the antibiotic susceptibility test. WGS data confirmed the identities of the isolates except Proteus vulgaris identified as P. terrae. More than one resistant determinant was detected on the draft genome sequence of 80% (8/10) of the randomly selected isolates especially the regulatory system modulating antibiotic efflux CRP and the plasmid mediated quinolone resistant determinant qnrD1. Significantly, one Proteus mirabilis isolate was sensitive to the antibiotics in the phenotypic testing but had resistance determinants present.&#x0D; Conclusion: This study provides genomic characterization of antibiotic resistant isolates from retail leafy and salad vegetables from Nigeria. Further study is important to understand the public health importance of such resistance and the amount of risk posed to human health by these resistant organisms.