Towards a better understanding of antimicrobial resistance dissemination: what can be learnt from studying model conjugative plasmids?

Bacteria can evolve rapidly by acquiring new traits such as virulence, metabolic properties, and most importantly, antimicrobial resistance, through horizontal gene transfer (HGT). Multidrug resistance in bacteria, especially in Gram-negative organisms, has become a global public health threat often through the spread of mobile genetic elements. Conjugation represents a major form of HGT and involves the transfer of DNA from a donor bacterium to a recipient by direct contact. Conjugative plasmids, a major vehicle for the dissemination of antimicrobial resistance, are selfish elements capable of mediating their own transmission through conjugation. To spread to and survive in a new bacterial host, conjugative plasmids have evolved mechanisms to circumvent both host defense systems and compete with co-resident plasmids. Such mechanisms have mostly been studied in model plasmids such as the F plasmid, rather than in conjugative plasmids that confer antimicrobial resistance (AMR) in important human pathogens. A better understanding of these mechanisms is crucial for predicting the flow of antimicrobial resistance-conferring conjugative plasmids among bacterial populations and guiding the rational design of strategies to halt the spread of antimicrobial resistance. Here, we review mechanisms employed by conjugative plasmids that promote their transmission and establishment in Gram-negative bacteria, by following the life cycle of conjugative plasmids.

Treatment of wound infections in a mouse model using Zn2+-releasing phage bound to gold nanorods

Infections caused by drug-resistant bacteria, particularly gram-negative organisms, are increasingly difficult to treat using antibiotics. A potential alternative is phage therapy, in which phages infect and lyse the bacterial host. However, phage therapy poses serious drawbacks and safety concerns, such as the risk of genetic transduction of antibiotic resistance genes, inconsistent pharmacokinetics, and unknown evolutionary potential. In contrast, metallic nanoparticles possess precise, tunable properties, including efficient conversion of electronic excitation into heat. In this work, we demonstrate that engineered phage-nanomaterial conjugates that target the gram-negative pathogen P. aeruginosa, are highly effective as a treatment of infected wounds in mice. Photothermal heating, performed as a single treatment (15 min) or as two treatments on consecutive days, rapidly reduced the bacterial load and released Zn2+ to promote wound healing. The phage-nanomaterial treatment was significantly more effective than systemic fluoroquinolone antibiotics in reducing both bacterial load and wound size, and was notably effective against a P. aeruginosa strain resistant to polymyxins, a last-line antibiotic therapy. Unlike these antibiotics, the phage-nanomaterial showed no detectable toxicity or systemic effects in mice, consistent with the short duration and localized nature of phage-nanomaterial treatment. Our results demonstrate that phage therapy controlled by inorganic nanomaterials can be a safe and effective antimicrobial strategy in vivo.

Antibiotic sensitivity and bacteriology of chronic suppurative otitis media in department of ENT in tertiary care centre St. John’s Medical College and Hospital Bangalore

<p><strong>Background: </strong>Chronic suppurative otitis media (CSOM) is a disease of middle ear. The aim of this study is to know the antibiotic sensitivity pattern in CSOM cases attending to ENT out patient department (OPD) in a tertiary care hospital and device an appropriate antibiotic protocol to treat CSOM cases.</p><p><strong>Methods: </strong>This is a prospective observational study. Fifty-eight patients of CSOM who present to the ENT OPD with ear discharge will be studied. Ear swabs of these patients will be collected and sent to microbiology to know the causative bacteria and their drug susceptibility.</p><p><strong>Results: </strong>Of the 58 patients studied 64% were males and 36% were females. Of the organisms isolated Stap aureus (53%) was most common followed by <em>Pseudomonas</em> (36%). All the gram-positive organisms were sensitive to cotimaxozole, amikacin and gentamycin and all gram-negative organisms were sensitive to ciprofloxacin, amikacin and netlimycin. </p><p><strong>Conclusions: </strong>It is better to collect an ear swab in all CSOM cases and then start antibiotics based upon the culture and sensitivity reports in order to improve the results and reduce the emergence of resistant strains, which can happen when antibiotics are prescribed randomly.</p>

Microbiological Profile of Neonatal Septicemia by Automated Blood Culture System (BacT/ALERT) at a Tertiary Care Hospital in Jamnagar, Gujarat, India

Background: This study was carried out to do rapid diagnosis of neonatal septicemia to prevent septicemia related complications and neonatal morbidity and mortality. Materials and Methods: This study was carried out in a tertiary care hospital from a period of March 2014 to August 2015. This is a non-randomized, prospective study in which one hundred and nine cases of suspected neonatal septicemia on the basis of antenatal high risk factors and signs and symptoms of sepsis were studied. Blood samples were collected from neonates in whom septicemia was suspected usually before antibacterial agents were given and both positive and negative samples were detected by automated system BacT/ALERT. Results: Out of 109 samples tested, 69 samples were positive and 40 samples were negative. The incidence of neonatal septicemia due to Gram positive organisms (62.85%) was found to be high. Among them Coagulase negative staphylococci (CONS) (27) (38.57%) was most common organism followed by Staphylococcus aureus (11) (15.71%), Enterococcus spp. (7.14%) and Streptococcus spp. (1.43%). Rate of Gram negative organisms was quite low (32.85%). Out of them Acinetobacter spp. (17.14%) was most common followed by Klebsiella spp. (7.14%), E.coli (5.71%) and Pseudomonas spp. (2.86%). Conclusion: Early detection of infection in neonates and proper antibiotics for the treatment leads to reduced morbidity and mortality.

Phenotypic characterization of carbapenem non-susceptible gram-negative bacilli isolated from clinical specimens

Background Multidrug resistant, extremely drug-resistant, pan-drug resistant, carbapenem-resistant, and carbapenemase-producing gram-negative bacteria are becoming more common in health care settings and are posing a growing threat to public health. Objective The study was aimed to detect and phenotypically characterize carbapenem no- susceptible gram-negative bacilli at the Ethiopian Public Health Institute. Materials and methods A prospective cross-sectional study was conducted from June 30, 2019, to May 30, 2020, at the national reference laboratory of the Ethiopian Public Health Institute. Clinical samples were collected, inoculated, and incubated for each sample in accordance with standard protocol. Antimicrobial susceptibility testing was conducted using Kirby-Bauer disk diffusion method. Identification was done using the traditional biochemical method. Multidrug-resistant and extensively drug-resistant isolates were classified using a standardized definition established by the European Centre for Disease Prevention and Control and the United States Centers for Disease Prevention and Control. Gram-negative organisms with reduced susceptibility to carbapenem antibiotics were considered candidate carbapenemase producers and subjected to modified carbapenem inactivation and simplified carbapenem inactivation methods. Meropenem with EDTA was used to differentiate metallo-β-lactamase (MBL) from serine carbapenemase. Meropenem (MRP)/meropenem + phenylboronic acid (MBO) were used to differentiate Klebsiella pneumoniae carbapenemase (KPC) from other serine carbapenemase producing gram-negative organisms. Results A total of 1,337 clinical specimens were analyzed, of which 429 gram-negative bacterial isolates were recovered. Out of 429 isolates, 319, 74, and 36 were Enterobacterales, Acinetobacter species, and Pseudomonas aeruginosa respectively. In our study, the prevalence of multidrug-resistant, extensively drug-resistant, carbapenemase-producing, and carbapenem nonsusceptible gram-negative bacilli were 45.2%, 7.7%, 5.4%, and 15.4% respectively. Out of 429 isolates, 66 demonstrated reduced susceptibility to the antibiotics meropenem and imipenem. These isolates were tested for carbapenemase production of which 34.8% (23/66) were carbapenemase producers. Out of 23 carbapenemase positive gram-negative bacteria, ten (10) and thirteen (13) were metallo-beta-lactamase and serine carbapenemase respectively. Three of 13 serine carbapenemase positive organisms were Klebsiella pneumoniae carbapenemase. Conclusion This study revealed an alarming level of antimicrobial resistance (AMR), with a high prevalence of multidrug-resistant (MDR) and extremely drug-resistant, carbapenemase-producing gram-negative bacteria, particularly among intensive care unit patients at the health facility level. These findings point to a scenario in which clinical management of infected patients becomes increasingly difficult and necessitates the use of “last-resort” antimicrobials likely exacerbating the magnitude of the global AMR crisis. This mandates robust AMR monitoring and an infection prevention and control program.

Gram staining v1

Gram staining is one of the first techniques used for the identification of group B Streptococcus agalactiae and one would expect to see gram-positive cocci under the microscope. The technique consists of applying a series of colorants and bleaches (acetone), which interact with the lipids of the membranes of gram-positive and gram-negative bacteria. The cell walls of gram-positive organisms retain the dye after acetone treatment and appear purple in color, whereas gram-negative organisms become discolored after acetone treatment and appear pink.

Investigator-Driven Randomised Controlled Trial of Cefiderocol versus Standard Therapy for Healthcare-Associated and Hospital-Acquired Gram-negative Bloodstream Infection: Study protocol (the GAME CHANGER trial): study protocol for an open-label, randomised controlled trial

Background Increasing rates of antibiotic resistance in Gram-negative organisms due to the presence of extended-spectrum beta-lactamases (ESBL), hyperproduction of AmpC enzymes, carbapenemases and other mechanisms of resistance are identified in common hospital- and healthcare-associated pathogens including Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii. Cefiderocol is a novel siderophore cephalosporin antibiotic with a catechol moiety on the 3-position side chain. Cefiderocol has been shown to be potent in vitro against a broad range of Gram-negative organisms, including carbapenem-resistant Enterobacteriaceae (CRE) and multi-drug-resistant (MDR) P. aeruginosa and A. baumannii. Recent clinical data has shown cefiderocol to be effective in the setting of complicated urinary tract infections and nosocomial pneumonia, but it has not yet been studied as treatment of bloodstream infection. Methods This study will use a multicentre, open-label non-inferiority trial design comparing cefiderocol and standard of care antibiotics. Eligible participants will be adult inpatients who are diagnosed with a bloodstream infection with a Gram-negative organism on the basis of a positive blood culture result where the acquisition meets the definition for healthcare-associated or hospital-acquired. It will compare cefiderocol with the current standard of care (SOC) antibiotic regimen according to the patient’s treating clinician. Eligible participants will be randomised 1:1 to cefiderocol or SOC and receive 5–14 days of antibiotic therapy. Trial recruitment will occur in at least 20 sites in ten countries (Australia, Malaysia, Singapore, Thailand, Turkey and Greece). The sample size has been derived from an estimated 14 day, all-cause mortality rate of 10% in the control group, and a non-inferiority margin of 10% difference in the two groups. A minimum of 284 patients are required in total to achieve 80% power with a two-sided alpha level of 0.05. Data describing demographic information, risk factors, concomitant antibiotics, illness scores, microbiology, multidrug-resistant organism screening, discharge and mortality will be collected. Discussion With increasing antimicrobial resistance, there is a need for the development of new antibiotics with broad activity against Gram-negative pathogens such as cefiderocol. By selecting a population at risk for multi-drug-resistant pathogens and commencing study treatment early in the clinical illness (within 48 h of index blood culture) the trial hopes to provide guidance to clinicians of the efficacy of this novel agent. Trial registration The GAME CHANGER trial is registered under the US National Institute of Health ClinicalTrials.gov register, reference number NCT03869437. Registered on March 11, 2019.

Acute Suppurative Thyroiditis Secondary to Escherichia coli Infection

Infections of the thyroid gland are rare. Its innate resistance to infections can be attributed to its unique anatomical features and rich blood supply. High clinical suspicion is required as a delay in diagnosis can lead to significant morbidity and mortality. Major pathogens include the Gram-positive Staphylococcus aureus and Streptococcus species; however, Gram-negative organisms have been found especially in immunocompromised hosts. We present a rare case of acute suppurative thyroiditis (AST) secondary to Escherichia coli (E. coli) infection in a woman known to be infected with human immunodeficiency virus (HIV).

Microbiological Profile of Uropathogens and their Antibiotic Susceptibility Pattern in Pediatric Population from A Tertiary Care Hospital in Amritsar, Punjab

Urinary tract infection(UTI) is one of the most commonest bacterial infection encountered in the pediatric age group. Early diagnosis is of utmost importance to preserve renal function and reduce long term complications such as renal scarring. The objective of this study was to determine the prevalence of UTI in pediatric population, identify the common uropathogens and study the antibiotic susceptibility pattern of bacterial isolates. Materials and Methods: A retrospective study was carried out in Department of Microbiology, GMC Amritsar for a period of 6 months from 1 Jan 2021 till 30 June 2021 amongst the pediatric patients (1 month-12 years age group) with clinically suspected UTI including both indoor and outdoor patients. Clean catch midstream urine samples received in the laboratory were processed as per the standard microbiological guidelines. Antibiotic susceptibility testing was done using Kirby-Bauer disc diffusion method as per CLSI guidelines. Results: A total of 549 samples were received and processed during study period from which 164(29.87%) were culture positive. Culture positivity was more prevalent in females (61.58%) than males (38.41%). Gram negative organisms were predominant (74.4%) followed by gram positive cocci (22.56%) and Candida (3.05%). Among gram negative organisms Escherichia coli was most predominant followed by Klebsiella spp., Pseudomonas spp., Acinetobacter spp., Proteus spp. and Citrobacter spp. Among gram positive cocci Staphylococcus aureus was most predominant followed by Coagulase negative Staphylococci and Enterococcus spp. Majority of gram negative bacilli were resistant to fluroquinolones, 3rd generation cephalosporins and co-trimoxazole while nitrofurantoin, piperacillin-tazobactam and gentamicin were most sensitive drugs. Among Staphylococcus aureus, methicillin resistance was seen in 17% isolates. High sensitivity was seen to linezolid and vancomycin in gram positive cocci. Conclusion: This study highlights increased prevalence of MDR uropathogens in pediatric population which indicates that antibiotic selection should be based on knowledge of local prevalence of bacterial organisms and their antibiotic sensitivities with rational use of antibiotics.

Neonatal Septicemia and Drug Resistance; 2 Year Prospective Study

Neonatal sepsis is a worldwide problem with the prevalence at 1 to10 per 1000 live birth and one of the indicator for measuring the health status of a nation. According to WHO there are about 5 million neonatal death per year with 98% occurring in developing countries. Antimicrobial resistance is a growing threat worldwide. Blood culture is considered as the gold standard technique for diagnosis of neonatal septicemia. The main objectives includes, to know the various bacteria causing neonatal septicemia and their antibiogram. To detect drug resistance among the isolated bacteria. A Prospective study was done over a period of 2 year. Blood samples for culture were collected aseptically before starting antibiotic therapy and subcultures were performed. The isolates were identified by standard biochemical tests and antibiogram of the isolates were studied. Out of 360 cases 160 were bacteriologically positive, Klebsiella was the most common organism isolated (21%), followed by Staphylococcus aureus (20%) Acinetobacter (15%), Pseudomonas (13%). Maximum sensitivity was seen by Linezolid, Erythromycin for Gram positive organisms and Gram negative organisms were sensitive to Piperacillin/ Tazobactum, Imipenem, Levofloxacin, Meropenem. Knowledge of likely causative organism causing neonatal septicemia can help in instituting prompt and appropriate therapy which in turn reduce morbidity and mortality.