Host biliary bacteria in cholecystectomy at a secondary-care teaching hospital, an observational study.

Background: Enterococcus faecalis rapidly develops resistance to different antibiotics, thereby resulting in serious nosocomial infections associated with high mortality rates and different problems in the healthcare systems. Objectives: This study aimed to analyze the genetic diversity, antimicrobial resistance, and virulence factors of E. faecalis isolates obtained from the stool samples of patients in a hospital in the center of Iran. Methods: In this cross-sectional descriptive-analytical study, 108 stool samples were collected from September 2019 to February 2020 from 108 patients hospitalized in a hospital in the center of Iran. Enterococcus faecalis isolates were detected using the ddlE gene detection technique, and antimicrobial resistance testing was performed using the disc agar diffusion method. Moreover, polymerase chain reaction was used to detect antimicrobial resistance genes and virulence factors. Genetic diversity was also analyzed by enterobacterial repetitive intergenic consensus using PCR. The BioNumerics software was used to construct a dendrogram. Results: Of 108 isolates, 50 samples were E. faecalis (46.2%). The prevalence of multidrug resistance among E. faecalis isolates was 62%, and most isolates were resistant to antibiotics tetracycline (70%), erythromycin (68%), and rifampin (60%). Among the E. faecalis isolates, the most prevalent antimicrobial resistance genes were ermB (96%), aph (2′′) Ia (66%), aac(6′)-Ie (40%), and ermC (30%), and the most prevalent virulence genes were gelE (78%), asa1 (74%), and esp (74%). The genetic diversity analysis showed 25 ERIC types in two major clusters (ie, clusters H and J) and eight minor clusters (ie, clusters A-G and I). There was no significant difference between clusters H and J in terms of antimicrobial resistance and resistance genes (P > 0.05). In contrast, the prevalence of the asa1 gene was significantly higher in cluster J than in cluster H (P < 0.05). Conclusions: This study showed the high prevalence of multidrug resistance, and high heterogeneity among the E. faecalis isolates obtained from the stool samples of hospitalized patients.

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. Therefore, rapid administration of adequate antibiotic therapy is of foremost importance in the treatment of bloodstream infections. The critical element in this process is timing, heavily dependent on the results of bacterial identification and antibiotic susceptibility testing. Both of these parameters are routinely obtained by culture-based testing, which is time-consuming and takes on average 24-48 hours2, 4. The aim of the study was to develop DNA-based assays for rapid identification of bloodstream infections, as well as rapid antimicrobial susceptibility testing. The first assay is a eubacterial 16S rDNA-based real-time PCR assay complemented with species- or genus-specific probes5. Using these probes, Gram-negative bacteria including Pseudomonas spp., Pseudomonas aeruginosa and Escherichia coli as well as Gram-positive bacteria including Staphylococcus spp., Staphylococcus aureus, Enterococcus spp., Streptococcus spp., and Streptococcus pneumoniae could be distinguished. Using this multiprobe assay, a first identification of the causative micro-organism was given after 2 h.Secondly, we developed a semi-molecular assay for antibiotic susceptibility testing of S. aureus, Enterococcus spp. and (facultative) aerobe Gram-negative rods6. This assay was based on a study in which PCR was used to measure the growth of bacteria7. Bacteria harvested directly from blood cultures are incubated for 6 h with a selection of antibiotics, and following a Sybr Green-based real-time PCR assay determines inhibition of growth. The combination of these two methods could direct the choice of a suitable antibiotic therapy on the same day (Figure 1). In conclusion, molecular analysis of both identification and antibiotic susceptibility offers a faster alternative for pathogen detection and could improve the diagnosis of bloodstream infections.

There are few studies comparing proportion, frequency, mortality and mortality rate following antimicrobial-resistant (AMR) infections between tertiary-care hospitals (TCHs) and secondary-care hospitals (SCHs) in low and middle-income countries (LMICs) to inform intervention strategies. The aim of this study is to demonstrate the utility of an offline tool to generate AMR reports and data for a secondary data analysis. We conducted a secondary-data analysis on a retrospective, multicentre data of hospitalised patients in Thailand. Routinely collected microbiology and hospital admission data of 2012 to 2015, from 15 TCHs and 34 SCHs were analysed using the AMASS v2.0 (www.amass.website). We then compared the burden of AMR bloodstream infections (BSI) between those TCHs and SCHs. Of 19,665 patients with AMR BSI caused by pathogens under evaluation, 10,858 (55.2%) and 8,807 (44.8%) were classified as community-origin and hospital-origin BSI, respectively. The burden of AMR BSI was considerably different between TCHs and SCHs, particularly of hospital-origin AMR BSI. The frequencies of hospital-origin AMR BSI per 100,000 patient-days at risk in TCHs were about twice that in SCHs for most pathogens under evaluation (for carbapenem-resistant Acinetobacter baumannii [CRAB]: 18.6 vs. 7.0, incidence rate ratio 2.77; 95%CI 1.72-4.43, p<0.001; for carbapenem-resistant Pseudomonas aeruginosa [CRPA]: 3.8 vs. 2.0, p = 0.0073; third-generation cephalosporin resistant Escherichia coli [3GCREC]: 12.1 vs. 7.0, p<0.001; third-generation cephalosporin resistant Klebsiella pneumoniae [3GCRKP]: 12.2 vs. 5.4, p<0.001; carbapenem-resistant K. pneumoniae [CRKP]: 1.6 vs. 0.7, p = 0.045; and methicillin-resistant Staphylococcus aureus [MRSA]: 5.1 vs. 2.5, p = 0.0091). All-cause in-hospital mortality (%) following hospital-origin AMR BSI was not significantly different between TCHs and SCHs (all p>0.20). Due to the higher frequencies, all-cause in-hospital mortality rates following hospital-origin AMR BSI per 100,000 patient-days at risk were considerably higher in TCHs for most pathogens (for CRAB: 10.2 vs. 3.6,mortality rate ratio 2.77; 95%CI 1.71 to 4.48, p<0.001; CRPA: 1.6 vs. 0.8; p = 0.020; 3GCREC: 4.0 vs. 2.4, p = 0.009; 3GCRKP, 4.0 vs. 1.8, p<0.001; CRKP: 0.8 vs. 0.3, p = 0.042; and MRSA: 2.3 vs. 1.1, p = 0.023). In conclusion, the burden of AMR infections in some LMICs might differ by hospital type and size. In those countries, activities and resources for antimicrobial stewardship and infection control programs might need to be tailored based on hospital setting. The frequency and in-hospital mortality rate of hospital-origin AMR BSI are important indicators and should be routinely measured to monitor the burden of AMR in every hospital with microbiology laboratories in LMICs.

Characteristics of third-generation cephalosporin-resistant Salmonella from retail chicken meat produced by integrated broiler operations

Aim: The aim was to study the occurrence of tetracycline resistance determinant tetA gene, and antibacterial resistance pattern in commensal Escherichia coli recovered from healthy non-clinical layer chickens. Materials and Methods: Twenty-four cloacal swabs were obtained from 15 flocks in five different layer farms located at around Namakkal, which is a place of high-intensity layer chicken rearing in south India. Identification of E. coli was carried out by performing cultural and biochemical tests. Antimicrobial resistance test was carried out using disc diffusion method. The polymerase chain reaction employing tetAC forward and tetAC reverse primers were carried out to detect tetA gene conferring resistance to tetracycline. Result: All the collected cloacal swabs yielded E. coli. Twenty-one isolates (88%) were resistant to tetracycline antibiotic in disc diffusion method. All the isolates showed resistance to more than six antibiotics, which implied existence of multidrug resistant microbiota in intestine of poultry. Only seven (29%) isolates showed the presence of tetA gene indicating the involvement of either other one or more efflux gene(s), namely tetB, tetC and tetD or ribosomal protection encoded by tetM, tetO, tetQ and tetS genes than tetA gene. Conclusion: Based on the presence of tetA gene among tetracycline-resistant bacteria in healthy non-clinical food-producing animals such as layer chickens, it can be significant in human medicine as tetA gene could easily be spread to other bacteria. This kind of phenomenon can be extrapolated in transfer of resistance for other antibacterial essential for treating bacterial infections in human. We conclude that preventing the spread of antimicrobial resistance through direct or indirect contact, consumed food/feed and through the environment is empirical in reduction of failures while treating bacterial infections.

Shigellosis is one of the major causes of morbidity in children with diarrhea in Iran. The aim of this study was to investigate antimicrobial resistance of Shigella flexneri (S. flexneri) strains isolated from clinically diagnosed cases of gastroen- teritis and acute diarrhea in Tehran, Iran. Shigella strains were isolated from stool samples of patients who visited the several major hospitals in Tehran. S. flexneri was preliminarily identified by biochemical tests as well as by API20E. Antimicrobial resistance testing was performed according to the standard guidelines of the Clinical and Laboratory Standards Institute. All strains were resistant to streptomycin. More than 96.4% of the strains were resistant to tetracycline and amoxicillin, 89% to co-trimoxsazole, 72.6% to ampicillin, 33.3% to chloramphenicol, 9.5% to kanamycin, 1.2% to cefixime, amikacin and furazoli- done. None of the tested isolates were resistant to ceftriaxone, ceftizoxime, ceftazidime, gentamicin, ciprofloxacin, cephalothin, cefotaxime, cephalexine, nalidixic acid and nitrofurantoin. More than 96% of the strains showed multi-drug resistance phe- notype. Seventeen resistance patterns were identified among the strains, however the most prevalent phenotype (36.9%) was R6 (streptomycin/amoxicillin/tetracycline/co-trimoxsazole/ampicillin). This study indicates an increase in incidence of multiple drug resistance among the strains of S. flexneri isolated in Tehran, Iran.

Background Prompt administration of antimicrobials improves sepsis outcomes. Molecular testing is transforming infectious disease diagnostics, but direct-from-blood identification of pathogens and antimicrobial resistance (AMR) has lagged. In this study, we tested the hypothesis that AMR genes could be identified in the peripheral blood of patients using RNA sequencing (RNA-seq). Methods A single center, prospective study was conducted of patients with sepsis in the intensive care unit (ICU) and with suspected infection in the emergency department (ED). Whole blood samples from ICU patients were drawn in PAXgene tubes on hospital day 0, 1, 3, and 7 and from ED patients at presentation. Deep RNA-seq of at least 100 million reads per sample was performed. Reads not mapped to human genome assembly GRCh38 (“unmapped” reads) were aligned to a custom AMR genome created from the Comprehensive Antibiotic Resistance Database (CARD). Command line bash and awk script and RStudio were utilized for computational processing. Results A total of 274 patients, 221 from the ED and 53 from the ICU, were included. This yielded 329 RNA-seq samples including data from multiple days, amounting to 3.3 billion reads. Of the 329 samples, 180 (54.7%) had no reads that aligned to the AMR genome while 149 (45.3%) had at least 2 reads that aligned. The 149 AMR-positive samples had a total of 3648 reads, with a median of 6 reads per sample (IQR: 12). When stratified by resistance mechanisms, most of the reads aligned to AMR genes coding for antibiotic efflux pumps (59.6%), followed by antibiotic inactivation (26.5%), target replacement (11.0%), target protection (2.2%), and reduced antibiotic permeability (0.7%). Conclusion Efflux pumps may be an underappreciated mechanism of AMR. Their expression leads to fitness costs, leading to tightly regulated (over)expression. This could cause a discordance between in vivo expression and in vitro where AMR testing is performed. More generally, RNA sequencing can identify AMR genes in peripheral blood, a dynamic assessment of expression since the half-life of RNA is short. Importantly, RNA will reflect AMR phenotypes better than DNA since mRNA is one step nearer to protein production. Disclosures Sean F. Monaghan, MD, Alcini, LLC: Ownership Interest

Invited Commentary

Antimicrobial Resistance (AMR) has emerged as a critical global health challenge, with bacteria, viruses, fungi, and parasites developing the capacity to survive antimicrobial treatments. This resistance, largely driven by increased antibiotic usage, threatens public health by diminishing the effectiveness of current infection management strategies. This study aims to evaluate the antimicrobial resistance patterns of prevalent pathogens in a secondary care hospital, highlighting the essential role of clinical pharmacists in addressing AMR through the implementation of Antibiotic Stewardship Programs (ASPs) to promote responsible antibiotic use. This prospective study analyzed 80 positive microbial culture reports from six months. Ethical approval was granted by the Institutional Ethical Committee (Ref: ECR/288/Indt/TN/2018/RR-21/001, dated April 6, 2023). Inclusion criteria covered adults (≥18 years) with confirmed infections across various sites, including bloodstream, urinary, respiratory, and soft tissue. Exclusion criteria eliminated reports with no pathogen growth. Data were analyzed using SPSS software version 26.0, with statistical measures applied to assess resistance patterns and correlations across infection types. Of the 80 positive cultures, Escherichia coli 35.0%) was most frequently isolated, followed by Klebsiella pneumoniae (12.5%), Pseudomonas aeruginosa (8.8%), Proteus mirabilis (8.8%), and Klebsiella oxytoca (7.5%). The isolated pathogens displayed high resistance to ampicillin (82.5%), cefixime (80.0%), ceftriaxone (78.8%), and ceftazidime (71.3%), with a strong sensitivity to amikacin (86.3%) and meropenem (70.0%). The rise of third-generation cephalosporin-resistant pathogens signals an urgent need for sustained AMR monitoring and robust ASPs in healthcare settings, particularly in developing regions. The study underscores the importance of rational antibiotic use and continuous AMR surveillance to curb resistant infections and protect public health.

Background: Knowledge about the treatment of skin and soft tissue infections in injecting drug users in countries with a low prevalence of antibiotic resistance is limited. We investigated bacterial antibiotic resistance and treatment of skin and soft tissue infections in Norwegian drug users.Methods: We performed a two year clinical cross-sectional observational study in a Norwegian hospital. Data were collected retrospectively from hospital records. We examined bacteriological findings and antibiotic resistance, and evaluated compliance to treatment guidelines and appropriateness of empirical antibiotic therapy relative to results of cultures and susceptibility testing. Descriptive and univariate analyses were performed.Results: Hundred and thirty-five injecting drug users were admitted with skin and soft tissue infection in the study period. Cultures were obtained from 103 (77%) abscesses and eight (24%) erysipelas and cellulitis, with bacterial growth in 80 (78%) and five (63%), respectively. Streptococci and staphylococci were the most prevalent bacteria, but methicillin-resistant Staphylococcus aureus was found in only one patient. Compliance to hospital antibiotic guidelines was 70%. Ninety-one per cent of patients in the compliant and 79% in the non-compliant group were given effective empirical antibiotics (p = .334). In the non-compliant group, significantly more patients received broad-spectrum empirical antibiotics (p < .001). In 30 cases where adjustment of antibiotic therapy was possible according to susceptibility testing, this was performed in only 14 cases.Conclusions: Bacteria and resistance patterns did not differ significantly from the skin and soft tissue infections in the general population in Norway. Compliance to antibiotic guidelines led to significantly less use of broad-spectrum antibiotics and to good bacterial coverage. General guidelines for treatment should be applied to injecting drug users with skin and soft tissue infections.

The demand for terrestrial snails as a food source is still on the increase globally, yet this has been overlooked in disease epidemiology and the spread of antimicrobial resistance. This study conducted genomic analyses of twenty Citrobacter portucalensis strains isolated from live edible snails traded in two hubs. The isolates were subjected to MALDI-TOF MS, antimicrobial resistance testing, whole genome sequencing, and analyses for in-depth characterization. The findings disclosed that seventeen strains across the two trading hubs were distinct from previously reported ones. Four isolates were found to share the same sequence type (ST881). Genome-based comparison suggests a clonal transmission of strains between snails traded in these hubs. All the isolates across the two hubs harbored similar variety of antimicrobial resistance genes, with notable ones being blaCMY and qnrB. Sixteen isolates (80%) expressed phenotypic resistance to second-generation cephalosporins, while eleven isolates (55%) exhibited resistance to third-generation cephalosporins. This report of multi-drug-resistant C. portucalensis strains in edible snails highlights significant concerns for food safety and clinical health because of the potential transmission to humans. Enhanced surveillance and stringent monitoring by health authorities are essential to evaluate the impact of these strains on the burden of antimicrobial resistance and to address the associated risk.

Methicillin-resistant Staphylococcus aureus (MRSA) have been found on German dairy farms and may be the cause of difficult-to-treat bovine mastitis. Considering the one health approach, MRSA might be transmitted from animals to humans raising the risk for severe infections. On 17 German dairy farms with a history of MRSA detection, MRSA strains were isolated from quarter milk, bulk tank milk, and swab samples of calves, heifers, pigs, and the environment. A selection of 33 isolates was analyzed using whole-genome sequencing and antimicrobial resistance testing. All detected MRSA strains were attributed to the livestock-associated sequence type 398. Methicillin-resistance was associated with the mecA gene in the staphylococcal cassette chromosome (SCC)mec types IVa (7/33) or V (26/33). The MRSA strains across the German federal states showed large allelic differences indicating independent development and distribution. On one farm, a clonal MRSA isolate was widely spread among different animals and the milking equipment. Moreover, MRSA transmission between two dairy farms in one federal state seems to be likely. In depth studies indicated that the resistance gene prediction and phenotypic resistance are in good agreement. Twenty eight strains were determined to exhibit a non-wildtype phenotype (resistant) against up to seven antimicrobial substances with an overall resistance to β-lactams and tetracycline. Ten different phenotypic antimicrobial resistance patterns were found among the MRSA strains. The strains harbored a wide virulence gene repertoire, of which some of them are related to bovine mastitis. However, the isolates lacked typical human infection associated factors such as the immune evasion cluster genes, staphylococcal enterotoxin genes, or Panton-Valentine leukocidin genes leading to the assumption for a low risk for severe human infections and foodborne diseases.

Prevalence, strain identification and antimicrobial resistance of Campylobacter spp. isolated from slaughtered pig carcasses in Brazil

Comprehensive evaluation of clinical antimicrobial resistance using impedance-accelerated single-bacterium multiplex screening strategy

In Uganda, the challenge of generating and timely reporting essential antimicrobial resistance (AMR) data has led to overreliance on empirical antibiotic therapy, exacerbating the AMR crisis. To address this issue, this study aimed to adapt a one-step AMR testing protocol alongside an SMS (Short Message Service) result relay system (SRRS), with the potential to reduce the turnaround time for AMR testing and result communication from 4 days or more to 1 day in Ugandan clinical microbiology laboratories. Out of the 377 samples examined, 54 isolates were obtained. Notably, E. coli (61%) and K. pneumoniae (33%) were the most frequently identified, majority testing positive for ESBL. Evaluation of three AMR testing protocols revealed varying sensitivity and specificity, with Protocol A (ChromID ESBL-based) demonstrating high sensitivity (100%) but no calculable specificity, Protocol B (ceftazidime-based) showing high sensitivity (100%) and relatively low specificity (7.1%), and Protocol C (cefotaxime-based) exhibiting high sensitivity (97.8%) but no calculable specificity. ESBL positivity strongly correlated with resistance to specific antibiotics, including cefotaxime, ampicillin, and aztreonam (100%), cefuroxime (96%), ceftriaxone (93%), and trimethoprim sulfamethoxazole (87%). The potential of integrating an SRRS underscored the crucial role this could have in enabling efficient healthcare communication in AMR management. This study underscores the substantial potential of the tested protocols for accurately detecting ESBL production in clinical samples, potentially, providing a critical foundation for predicting and reporting AMR patterns. Although considerations related to specificity warrant careful assessment before widespread clinical adoption.