Prevalence and antimicrobial resistance pattern of Clostridium difficile among hospitalized diarrheal patients: A systematic review and meta-analysis

Background Clostridium difficile is the leading cause of infectious diarrhea that develops in patients after hospitalization during antibiotic administration. It has also become a big issue in community-acquired diarrhea. The emergence of hypervirulent strains of C. difficile poses a major problem in hospital-associated diarrhea outbreaks and it is difficult to treat. The antimicrobial resistance in C. difficile has worsened due to the inappropriate use of broad-spectrum antibiotics including cephalosporins, clindamycin, tetracycline, and fluoroquinolones together with the emergence of hypervirulent strains. Objective To estimate the pooled prevalence and antimicrobial resistance pattern of C. difficile derived from hospitalized diarrheal patients, a systematic review and meta-analysis was performed. Methods Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guideline was followed to review published studies conducted. We searched bibliographic databases from PubMed, Scopus, Google Scholar, and Cochrane Library for studies on the prevalence and antimicrobial susceptibility testing on C. difficile. The weighted pooled prevalence and resistance for each antimicrobial agent was calculated using a random-effects model. A funnel plot and Egger’s regression test were used to see publication bias. Results A total of 15 studies were included. Ten articles for prevalence study and 5 additional studies for antimicrobial susceptibility testing of C. difficile were included. A total of 1967/7852 (25%) C. difficile were isolated from 10 included studies for prevalence study. The overall weighted pooled proportion (WPP) of C. difficile was 30% (95% CI: 10.0–49.0; p<0.001). The analysis showed substantial heterogeneity among studies (Cochran’s test = 7038.73, I2 = 99.87%; p<0.001). The weighed pooled antimicrobial resistance (WPR) were: vancomycin 3%(95% CI: 1.0–4.0, p<0.001); metronidazole 5%(95% CI: 3.0–7.0, p<0.001); clindamycin 61%(95% CI: 52.0–69.0, p<0.001); moxifloxacin 42%(95% CI: 29–54, p<0.001); tetracycline 35%(95% CI: 22–49, p<0.001); erythromycin 61%(95% CI: 48–75, p<0.001) and ciprofloxacin 64%(95% CI: 48–80; p< 0.001) using the random effect model. Conclusions A higher weighted pooled prevalence of C. difficile was observed. It needs a great deal of attention to decrease the prevailing prevalence. The resistance of C. difficile to metronidazole and vancomycin was low compared to other drugs used to treat C. difficile infection. Periodic antimicrobial resistance monitoring is vital for appropriate therapy of C. difficile infection.

Genomic evolution of the globally disseminated multidrug-resistant Klebsiella pneumoniae clonal group 147

The rapid emergence of multidrug-resistant Klebsiella pneumoniae is being driven largely by the spread of specific clonal groups (CGs). Of these, CG147 includes 7-gene multilocus sequence typing (MLST) sequence types (STs) ST147, ST273 and ST392. CG147 has caused nosocomial outbreaks across the world, but its global population dynamics remain unknown. Here, we report a pandrug-resistant ST147 clinical isolate from India (strain DJ) and define the evolution and global emergence of CG147. Antimicrobial-susceptibility testing following European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines and genome sequencing (Illumina and Oxford Nanopore Technologies, Unicycler assembly) were performed on strain DJ. Additionally, we collated 217 publicly available CG147 genomes [National Center for Biotechnology Information (NCBI), May 2019]. CG147 evolution was inferred within a temporal phylogenetic framework (beast) based on a recombination-free sequence alignment (Roary/Gubbins). Comparative genomic analyses focused on resistance and virulence genes and other genetic elements (BIGSdb, Kleborate, PlasmidFinder, phaster, ICEfinder and CRISPRCasFinder). Strain DJ had a pandrug-resistance phenotype. Its genome comprised the chromosome, seven plasmids and one linear phage-plasmid. Four carbapenemase genes were detected: bla NDM-5 and two copies of bla OXA-181 in the chromosome, and a second copy of bla NDM-5 on an 84 kb IncFII plasmid. CG147 genomes carried a mean of 13 acquired resistance genes or mutations; 63 % carried a carbapenemase gene and 83 % harboured bla CTX-M. All CG147 genomes presented GyrA and ParC mutations and a common subtype I-E CRISPR-Cas system. ST392 and ST273 emerged in 2005 and 1995, respectively. ST147, the most represented phylogenetic branch, was itself divided into two main clades with distinct capsular loci: KL64 (74 %, DJ included, emerged in 1994 and disseminated worldwide, with carbapenemases varying among world regions) and KL10 (20 %, emerged in 2002, predominantly found in Asian countries, associated with carbapenemases NDM and OXA-48-like). Furthermore, subclades within ST147-KL64 differed at the yersiniabactin locus, OmpK35/K36 mutations, plasmid replicons and prophages. The absence of IncF plasmids in some subclades was associated with a possible activity of a CRISPR-Cas system. K. pneumoniae CG147 comprises pandrug-resistant or extensively resistant isolates, and carries multiple and diverse resistance genes and mobile genetic elements, including chromosomal bla NDM-5. Its emergence is being driven by the spread of several phylogenetic clades marked by their own genomic features and specific temporo–spatial dynamics. These findings highlight the need for precision surveillance strategies to limit the spread of particularly concerning CG147 subsets.

Emergence and Genomic Characterization of a KPC-2-, NDM-1-, and IMP-4-Producing Klebsiella michiganensis Isolate

A rectal swab sample was collected from a patient with Guillain–Barré syndrome and enriched in lysogeny broth. Carbapenem-resistant bacteria were selected by China Blue agar plates containing 0.3 μg/ml meropenem. Carbapenemase-producing Klebsiella michiganensis was identified and characterized by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF), immune colloidal gold technique, a conjugation experiment, PCR analysis, and antimicrobial susceptibility testing. The genome of K. michiganensis was determined by whole genome sequencing. Antimicrobial susceptibility testing showed that the K. michiganensis was resistant to imipenem, meropenem, ertapenem, cefmetazole, ceftazidime, cefotaxime, piperacillin/tazobactam, sulbactam/cefoperazone, ceftazidime/avibactam, cefepime, and aztreonam while susceptible to polymyxin B, ciprofloxacin, tigecycline, and amikacin. Immune colloidal gold technique suggested that this strain co-produced three different carbapenemases [Klebsiella pneumoniae carbapenemase (KPC), New Delhi metallo-beta-lactamase (NDM), and Imipenem (IMP)]. Whole genome sequencing analysis indicated that this strain belonged to ST91, and blaKPC–2, blaNDM–1, and blaIMP–4 were carried on different conjugative plasmids. Besides, the co-existence and transferability of blaKPC–2, blaNDM–1, and blaIMP–4 in K. michiganensis facilitates the potential horizontal dissemination and nosocomial spread of resistance genes among multidrug-resistant organisms.

Real Life Clinical Impact of Antimicrobial Stewardship Actions on the Blood Culture Workflow from a Microbiology Laboratory

Background: Accelerating the diagnosis of bacteremia is one of the biggest challenges in clinical microbiology departments. The fast establishment of a correct treatment is determinant on bacteremic patients’ outcomes. Our objective was to evaluate the impact of antimicrobial therapy and clinical outcomes of a rapid blood culture workflow protocol in positive blood cultures with Gram-negative bacilli (GNB). Methods: A quasi-experimental before–after study was performed with two groups: (i) control group (conventional work-protocol) and (ii) intervention group (rapid workflow-protocol: rapid identification by Matrix-Assisted Laser Desorption/Ionization-Time-Of-Flight (MALDI-TOF) and antimicrobial susceptibility testing (AST) from bacterial pellet without overnight incubation). Patients were divided into different categories according to the type of intervention over treatment. Outcomes were compared between both groups. Results: A total of 313 patients with GNB-bacteremia were included: 125 patients in the control group and 188 in the intervention. The time from positive blood culture to intervention on antibiotic treatment decreased from 2.0 days in the control group to 1.0 in the intervention group (p < 0.001). On the maintenance of correct empirical treatment, the control group reported 2.0 median days until the clinical decision, while in the intervention group was 1.0 (p < 0.001). In the case of treatment de-escalation, a significant difference between both groups (4.0 vs. 2.0, p < 0.001) was found. A decreasing trend on the change from inappropriate treatments to appropriate ones was observed: 3.5 vs. 1.5; p = 0.12. No significant differences were found between both groups on 7-days mortality or on readmissions in the first 30-days. Conclusions: Routine implementation of a rapid workflow protocol anticipates the report of antimicrobial susceptibility testing results in patients with GNB-bacteremia, decreasing the time to effective and optimal antibiotic therapy.

Antibiotic Resistance among Escherichia coli Isolates from Hospital Wastewater Compared to Community Wastewater

This study aimed to compare the antibiotic resistance levels of the indicator bacteria Escherichia coli in wastewater samples collected from two hospitals and two urban communities. Antimicrobial susceptibility testing was performed on 81 E. coli isolates (47 from hospitals and 34 from communities) using the disc diffusion method according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) methodology. Ten antibiotics from nine different classes were chosen. The strains isolated from the community wastewater, compared to those from the hospital wastewater, were not resistant to gentamicin (p = 0.03), but they showed a significantly higher susceptibility—increased exposure to ceftazidime (p = 0.001). Multidrug resistance was observed in 85.11% of the hospital wastewater isolates and 73.53% of the community isolates (p > 0.05). The frequency of the presumed carbapenemase-producing E. coli was higher among the community isolates (76.47% compared to 68.09%) (p > 0.05), whereas the frequency of the presumed extended-spectrum beta-lactamase (ESBL)-producing E. coli was higher among the hospital isolates (21.28% compared to 5.88%) (p > 0.05). The antibiotic resistance rates were high in both the hospital and community wastewaters, with very few significant differences between them, so the community outlet might be a source of resistant bacteria that is at least as important as the well-recognised hospitals.

Comparison of novel rapid diagnostic of blood culture identification and antimicrobial susceptibility testing by Accelerate Pheno system and BioFire FilmArray Blood Culture Identification and BioFire FilmArray Blood Culture Identification 2 panels

Background Conventional turnaround time (TAT) for positive blood culture (PBC) identification (ID) and antimicrobial susceptibility testing (AST) is 2–3 days. We evaluated the TAT and ID/AST performance using clinical and seeded samples directly from PBC bottles with different commercial approaches: (1) Accelerate Pheno® system (Pheno) for ID/AST; (2) BioFire® FilmArray® Blood Culture Identification (BCID) Panel and/ or BCID2 for ID; (3) direct AST by VITEK® 2 (direct AST); and (4) overnight culture using VITEK® 2 colony AST. Results A total of 141 PBC samples were included in this evaluation. Using MALDI-TOF (Bruker MALDI Biotyper) as the reference method for ID, the overall monomicrobial ID sensitivity/specificity are as follows: Pheno 97.9/99.9%; BCID 100/100%; and BCID2 100/100%, respectively. For AST performance, broth microdilution (BMD) was used as the reference method. For gram-negatives, overall categorical and essential agreements (CA/EA) for each method were: Pheno 90.3/93.2%; direct AST 92.6/88.5%; colony AST 94.4/89.5%, respectively. For gram-positives, the overall CA/EAs were as follows: Pheno 97.2/98.89%; direct AST 97.2/100%; colony AST 97.2/100%, respectively. The BCID/BCID2 and direct AST TATs were around 9–20 h (1/9-19 h for ID with resistance markers/AST), with 15 min/sample hands-on time. In comparison, Pheno TATs were around 8–10 h (1.5/7 h for ID/AST) with 2 min/sample hands-on time, maintains a clinically relevant fast report of antibiotic minimal inhibitory concentration (MIC) and allows for less TAT and hands-on time. Conclusion In conclusion, to the best of our knowledge, this is the first study conducted as such in Asia; all studied approaches achieved satisfactory performance, factors such as TAT, panel of antibiotics choices and hands-on time should be considered for the selection of appropriate rapid ID and AST of PBC methods in different laboratory settings.

Development of an In-House Rapid Antimicrobial Susceptibility Testing Protocol for Positive Blood Culture and Its Implementation in Routine Microbiology Laboratories

Bloodstream infections (BSI) are associated with high morbidity and mortality and remain a leading cause of death. Blood culture (BC) including the identification and the antimicrobial susceptibility testing of the causative microorganisms should be performed as soon as possible. In this study, we developed an in-house rapid antimicrobial susceptibility testing (rAST) protocol for positive BC. First, the rAST was performed in the simulated positive BC of standard strains (Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, and Pseudomonas aeruginosa ATCC 27853) at three different times to assess the reproducibility and operability by dispensing four drops of BC broth onto a Mueller–Hinton agar plate after a positive signal. Furthermore, the rAST was performed in clinical positive BCs. The results of rAST at 4, 6, 8, and 18 h of incubation were compared with results of the standard 16- to 20-h disk diffusion method, and the preliminary breakpoints of the rAST method were established according to the inhibition diameter of sensitive strains and resistant strains. Finally, the rAST was performed in the simulated positive BC of clinical strains to evaluate the availability of the preliminary breakpoints. The rAST results of standard strains were distributed evenly at three different times. Among the 202 clinical strains used to establish the preliminary breakpoints, the number of zone diameters that could be read and interpreted (60, 87, 98, and 100%) increased with incubation time (4, 6, 8, and 18 h), and the categorical agreement was acceptable, with total error rates of 3.0, 2.3, 2.1, and 1.3% at 4, 6, 8, and 18 h of incubation, respectively. In conclusion, the in-house rAST protocol for positive BC can be implemented in routine laboratories. It provides reliable antimicrobial susceptibility testing results for BSI pathogens after 4–6 h of incubation.