Review of the Impact of Biofilm Formation on Recurrent Clostridioides difficile Infection.

Clostridioides difficile infection (CDI) may recur in approximately 10-30% of patients, and the risk of recurrence increases with each successive recurrence, reaching up to 65%. C. difficile can form biofilm with approximately 20% of the bacterial genome expressed differently between biofilm and planktonic cells. Biofilm plays several roles that may favor recurrence; for example, it may act as a reservoir of spores, protect the vegetative cells from the activity of antibiotics, and favor the formation of persistent cells. Moreover, the expression of several virulence genes, including TcdA and TcdB toxins, has been associated with recurrence. Several systems and structures associated with adhesion and biofilm formation have been studied in C. difficile, including cell-wall proteins, quorum sensing (including LuxS and Agr), Cyclic di-GMP, type IV pili, and flagella. Most antibiotics recommended for the treatment of CDI do not have activity on spores and do not eliminate biofilm. Therapeutic failure in R-CDI has been associated with the inadequate concentration of drugs in the intestinal tract and the antibiotic resistance of a biofilm. This makes it challenging to eradicate C. difficile in the intestine, complicating antibacterial therapies and allowing non-eliminated spores to remain in the biofilm, increasing the risk of recurrence. In this review, we examine the role of biofilm on recurrence and the challenges of treating CDI when the bacteria form a biofilm.

Transcriptomic analysis of biofilm formation in strains of Clostridioides difficile associated with recurrent and non-recurrent infection reveals potential candidate markers for recurrence.

The transcriptomic profile in a biofilm model of ribotypes (RT) 001 and 027 associated with recurrent Clostridioides difficile infection (R-CDI) and not associated with recurrent (NR)-CDI was analyzed to identify genes that may favor the recurrence. Twenty strains were selected, 10 RT001 and 10 RT027. From each ribotype, 5 were R-CDI and 5 NR-CDI. Biofilm and nonadherent cells were prepared from each clinical isolate, and the RNA was extracted. RNA samples were pooled in 8 combinations implying ribotype, recurrence, and biofilm formation. Each pool was separately labeled with Cy3 dye and hybridized on a microarray designed for this study. Slides were scanned, analyzed, and gene expression was compared between unique and grouped pools using the Student's t-test with Benjamini-Hochberg correction when appropriate. Validation was carried out by qRT-PCR for selected genes. Results: After comparisons of differentially expressed genes from both ribotypes of R-CDI strains (nonadherent cells vs. biofilm) and both ribotypes in biofilm (R-CDI vs. NR-CDI), we found 3 genes over-expressed and 1 under-expressed in common (adj. p ≤ 0.05). Overexpressed genes were CAJ70148 (a putative dehydrogenase), CAJ68100 (a secretion type II system protein from the GspH (pseudopilins) family), and CAJ69725 (a putative membrane protein); under-expressed was CAJ68151 (a segregation and condensation protein A). Because CAJ70148, CAJ68100, CAJ69725 and CAJ68151 were differentially expressed in biofilm in strains associated with R-CDI, they may support the biofilm favoring the recurrence of CDI. However, further studies will be needed for poorly studied genes.

How Does Hospital Microbiota Contribute to Healthcare-Associated Infections?

Healthcare-associated infections (HAIs) are still a global public health concern, associated with high mortality and increased by the phenomenon of antimicrobial resistance. Causative agents of HAIs are commonly found in the hospital environment and are monitored in epidemiological surveillance programs; however, the hospital environment is a potential reservoir for pathogenic microbial strains where microorganisms may persist on medical equipment surfaces, on the environment surrounding patients, and on corporal surfaces of patients and healthcare workers (HCWs). The characterization of hospital microbiota may provide knowledge regarding the relatedness between commensal and pathogenic microorganisms, their role in HAIs development, and the environmental conditions that favor its proliferation. This information may contribute to the effective control of the dissemination of pathogens and to improve infection control programs. In this review, we describe evidence of the contribution of hospital microbiota to HAI development and the role of environmental factors, antimicrobial resistance, and virulence factors of the microbial community in persistence on hospital surfaces.

Analysis of biofilm production and expression of adhesion structures of circulating Clostridioides difficile strains from Mexico.

INTRODUCTION: Clostridioides difficile biofilms are believed to protect the pathogen from antibiotics, in addition to potentially contributing to recurrent infections. METHODOLOGY: Biofilm production of 102 C. difficile isolates was determined using the crystal violet staining technique, and detachment assays were performed. The expression levels of cwp84 and slpA genes were evaluated by real-time PCR on selected isolates. RESULTS: More than 70% of isolates (75/102) were strong biofilm producers, and the highest detachment of biofilm was achieved with the proteinase K treatment (>90%). The overall mean expression of cwp84 was higher in RT027 than in RT001 (p=0.003); among strong biofilm-producing strains, the slpA expression was lower in RT027 than in RT001 (p<0.000). CONCLUSIONS: Proteins seem to have an important role in the biofilm's initial adherence and maturation. slpA and cwp84 are differentially expressed by C. difficile ribotype and biofilm production level.

Analysis of biofilm production and expression of adhesion structures of circulating Clostridioides difficile strains from Mexico / Análisis de la producción de biopelícula y expresión de estructuras de adhesión de cepas circulantes de Clostridioides difficile en México

Introduction: Clostridioides difficile biofilms are believed to protect the pathogen from antibiotics, in addition to potentially contributing to recurrent infections. Methodology: Biofilm production of 102 C. difficile isolates was determined using the crystal violet staining technique, and detachment assays were performed. The expression levels of cwp84 and slpA genes were evaluated by real-time PCR on selected isolates. Results: More than 70% of isolates (75/102) were strong biofilm producers, and the highest detachment of biofilm was achieved with the proteinase K treatment (>90%). The overall mean expression of cwp84 was higher in RT027 than in RT001 (p=0.003); among strong biofilm-producing strains, the slpA expression was lower in RT027 than in RT001 (p<0.000). Conclusions: Proteins seem to have an important role in the biofilm's initial adherence and maturation. slpA and cwp84 are differentially expressed by C. difficile ribotype and biofilm production level.(AU)

Introducción: Se cree que las biopelículas de Clostridioides difficile (C. difficile) protegen al patógeno de los antibióticos, además de contribuir potencialmente a las infecciones recurrentes. Metodología: Se determinó la producción de biopelículas de 102 aislados de C. difficile, mediante la técnica de tinción con violeta cristal y se realizaron ensayos de desprendimiento. Los niveles de expresión de los genes cwp84 y slpA se evaluaron mediante PCR en tiempo real en aislados seleccionados. Resultados: Más del 70% de los aislados (75/102) fueron fuertes productores de biopelículas y el mayor desprendimiento de biopelícula se logró con el tratamiento con proteinasa K (> 90%). La expresión media global de cwp84 fue mayor en RT027 que en RT001 (p = 0,003); entre las cepas productoras fuertes de biopelícula, la expresión de slpA fue menor en RT027 que en RT001 (p < 0,000). Conclusiones: Las proteínas parecen tener un papel importante en la adhesión y maduración inicial de las biopelículas; slpA y cwp84 se expresan de forma diferente según el ribotipo de C. difficile y el nivel de producción de biopelículas.(AU)

Contamination of healthcare environment by carbapenem-resistant Acinetobacter baumannii.

Acinetobacter baumannii is frequently found on floors, devices, and environmental sites in hospitals and can survive for prolonged periods and accumulate resistance determinants. The infection and presence of carbapenem-resistant A. baumannii (CRAB) in patients is associated with increased mortality, severe clinical outcomes, and longer lengths of stay at hospitals. This review addresses contamination by CRAB in corporal surfaces of patients and healthcare workers and environmental sites at healthcare-related settings. We summarized published data during the last decade on potential reservoirs for CRAB, including contamination frequency and the involved resistance mechanisms, and some measures associated with the elimination of CRAB from hospital surfaces.

Efficacy and In Vitro Activity of Novel Antibiotics for Infections With Carbapenem-Resistant Gram-Negative Pathogens.

Infections by Gram-negative multi-drug resistant (MDR) bacterial species are difficult to treat using available antibiotics. Overuse of carbapenems has contributed to widespread resistance to these antibiotics; as a result, carbapenem-resistant Enterobacterales (CRE), A. baumannii (CRAB), and P. aeruginosa (CRPA) have become common causes of healthcare-associated infections. Carbapenems, tigecycline, and colistin are the last resource antibiotics currently used; however, multiple reports of resistance to these antimicrobial agents have been documented worldwide. Recently, new antibiotics have been evaluated against Gram-negatives, including plazomicin (a new aminoglycoside) to treat CRE infection, eravacycline (a novel tetracycline) with in vitro activity against CRAB, and cefiderocol (a synthetic conjugate) for the treatment of nosocomial pneumonia by carbapenem-non-susceptible Gram-negative isolates. Furthermore, combinations of known ß-lactams with recently developed ß-lactam inhibitors, such as ceftazidime-avibactam, ceftolozane-tazobactam, ceftazidime-tazobactam, and meropenem-vaborbactam, has been suggested for the treatment of infections by extended-spectrum ß-lactamases, carbapenemases, and AmpC producer bacteria. Nonetheless, they are not active against all carbapenemases, and there are reports of resistance to these combinations in clinical isolates.This review summarizes and discusses the in vitro and clinical evidence of the recently approved antibiotics, ß-lactam inhibitors, and those in advanced phases of development for treating MDR infections caused by Gram-negative multi-drug resistant (MDR) bacterial species.

An Update on Clostridioides difficile Binary Toxin.

Infection with Clostridioides difficile (CDI), a common healthcare-associated infection, includes symptoms ranging from mild diarrhea to severe cases of pseudomembranous colitis. Toxin A (TcdA) and toxin B (TcdB) cause cytotoxicity and cellular detachment from intestinal epithelium and are responsible for CDI symptomatology. Approximately 20% of C. difficile strains produce a binary toxin (CDT) encoded by the tcdA and tcdB genes, which is thought to enhance TcdA and TcdB toxicity; however, the role of CDT in CDI remains controversial. Here, we focused on describing the main features of CDT and its impact on the host, clinical relevance, epidemiology, and potential therapeutic approaches.

Clinical characteristics associated with the severity of Clostridium [Clostridioides] difficile infection in a tertiary teaching hospital from Mexico.

BACKGROUND: Clostridium difficile infection (CDI) is a leading cause of healthcare-associated diarrhea worldwide. In this study, risk factors associated with the development of severe-complicated and recurrent outcomes in CDI patients in different age groups, including the non-elderly, were assessed in a third-level hospital. METHODS: CDI cases were detected by clinical data and polymerase-chain-reaction (PCR). Clinical, demographic, epidemiological, and microbiological risk factors for CDI were evaluated. RESULTS: During the study period, 248 out of 805 patients with nosocomial diarrhea were diagnosed with CDI and the majority were severe-complicated cases (87.90%). Female gender (OR 3.19, 95% CI 1.19-8.55, p = 0.02) and lymphoma (OR 3.95, 95% CI 1.03-15.13, p = 0.04) were risk factors for severe-complicated CDI. Mature adulthood (51-60 years) (OR 5.80, 95% CI 1.56-21.62, p = 0.01), previous rifampicin use (OR 7.44, 95% CI 2.10-26.44, p = 0.00), and neoplasm (solid malignant neoplasm or hematological malignancies) (OR 4.12, 95% CI 1.01-16.83, p = 0.04) were risk factors for recurrent infection. Autoimmune disorders (OR 6.62, CI 95% 1.26-34.73, p = 0.02), leukemia (OR 4.97, 95% CI 1.05-23.58, p = 0.04), lymphoma (OR 3.79, 95% CI 1.03-12.07, p = 0.04) and previous colistin treatment (OR 4.97, 95% CI 1.05-23.58, p = 0.04) were risk factors for 30-day mortality. CONCLUSION: Newly identified risk factors for recurrent CDI were rifampicin treatment and age between 51 and 60 years; colistin treatment was identified as a risk factor for 30-day mortality. Previously identified risk factors for severe-complicated CDI were confirmed, but with a major impact on non-elderly patients.

Diversity of Circulating Clostridioides difficile Ribotypes in Mexico and Susceptibility to Fidaxomicin, Vancomycin, and Metronidazole.

In this study, we report the results of the epidemiological analysis of Clostridioides difficile ribotypes (RTs) and antimicrobial susceptibility testing. Most isolates were RT027, representing 73% (84/115) of isolates. No isolates with reduced susceptibility to fidaxomicin were found; however, 38 (33.04%) isolates had reduced susceptibility to metronidazole, and 7 isolates (6.1%) had reduced susceptibility to vancomycin. These findings highlight the need for continuous surveillance of C. difficile RTs and antimicrobial susceptibility testing.

Analysis of biofilm production and expression of adhesion structures of circulating Clostridioides difficile strains from Mexico.

INTRODUCTION: Clostridioides difficile biofilms are believed to protect the pathogen from antibiotics, in addition to potentially contributing to recurrent infections. METHODOLOGY: Biofilm production of 102 C. difficile isolates was determined using the crystal violet staining technique, and detachment assays were performed. The expression levels of cwp84 and slpA genes were evaluated by real-time PCR on selected isolates. RESULTS: More than 70% of isolates (75/102) were strong biofilm producers, and the highest detachment of biofilm was achieved with the proteinase K treatment (>90%). The overall mean expression of cwp84 was higher in RT027 than in RT001 (p=0.003); among strong biofilm-producing strains, the slpA expression was lower in RT027 than in RT001 (p<0.000). CONCLUSIONS: Proteins seem to have an important role in the biofilm's initial adherence and maturation. slpA and cwp84 are differentially expressed by C. difficile ribotype and biofilm production level.

Microbial diversity and colonization patterns of two step-down care units from a tertiary care hospital.

Nosocomial surfaces are potential pathogen reservoirs. Our aim was to describe the microbial diversity and analyze microbial patterns of healthcare-associated pathogens in two step-down-care-units at a tertiary care hospital. We monitored infected patients over 45 days to describe microbial diversity and colonization patterns. A total of 2762 isolates were recovered from the sampled sites, coagulase-negative staphylococci represented 44.64% (1233/2762) of the isolates. The most frequently recovered ESKAPE species (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter cloacae) were A. baumannii (7.53%; 208/2762 isolates) and E. faecium/Enterococcus faecalis (5.18%; 143/2762). We recovered a high diversity of species, including potential pathogens. A. baumannii was detected more frequently on diverse surfaces and persisted in patients' nostrils during the hospital stay.

Molecular epidemiology of predominant and emerging Clostridioides difficile ribotypes.

There has been an increase in the incidence and severity of Clostridioides difficile infection (CDI) worldwide, and strategies to control, monitor, and diminish the associated morbidity and mortality have been developed. Several typing methods have been used for typing of isolates and studying the epidemiology of CDI; serotyping was the first typing method, but then was replaced by pulsed-field gel electrophoresis (PFGE). PCR ribotyping is now the gold standard method; however, multi locus sequence typing (MLST) schemes have been developed. New sequencing technologies have allowed comparing whole bacterial genomes to address genetic relatedness with a high level of resolution and discriminatory power to distinguish between closely related strains. Here, we review the most frequent C. difficile ribotypes reported worldwide, with a focus on their epidemiology and genetic characteristics.

Dynamics of colonization in patients with health care-associated infections at step-down care units from a tertiary care hospital in Mexico.

BACKGROUND: Patient colonization has been suggested as a risk factor in hospital-associated infections (HAI) development, which are of the most frequent complications in hospitals. OBJECTIVE: To examine the colonization process and possible transmission routes of HAI-causative agents in step-down care unit (SDCU) patients. METHODS: Patients admitted to SDCU within 48 hours of admission that had no evidence of infection present, nurse health care workers (HCWs), and relatives of infected patients were included. Participants were sampled and cultured at different times in different body surfaces. Environmental surfaces and medical devices were also sampled. Antimicrobial susceptibility and clonal relatedness were determined in selected HAI-causative agents, environmental, nurse HCWs, and patient isolates. RESULTS: A total of 2,735 isolates corresponding to 126 species were identified. Of the 11 patients included, 8 developed 1-3 HAIs (14 isolates recovered as HAI-causative agents). Acinetobacter baumannii (36% of infections) was distributed in clone A (n = 1), B (n = 3), and F (n = 1); Klebsiella pneumoniae (29%) in clones A (n = 2) and B (n = 1) and Enterobacter cloacae (7%) in one clone A. Causative agents were progressively recovered from environmental surfaces and medical devices before and after HAI onset. CONCLUSIONS: Highly related strains were recovered from environmental surfaces, patients, and nurse HCWs before and after HAI outcome. This is a first step to examine colonization process in SDCU settings and provides a base for further studies to understand colonization dynamics and the role of patients' relatives and nurse HCWs in organism transmission in the SDCU.

Screening of biomarkers of drug resistance or virulence in ESCAPE pathogens by MALDI-TOF mass spectrometry.

Rapid identification and characterisation of drug-resistant bacterial pathogens have an important role in diagnostic and antimicrobial stewardship. Response time in the diagnosis of not only the etiological agent but also in antimicrobial susceptibility results is of utmost importance in patient treatment. In this study, matrix-assisted laser desorption ionisation-time of flight (MALDI-TOF) mass spectrometry (MS) was used to screen for biomarkers of ESCAPE (vancomycin-resistant Enterococcus faecium, methicillin-resistant Staphylococcus aureus, hypervirulent NAP1/ribotype 027 Clostridioides [Clostridium] difficile, multidrug resistant Acinetobacter baumannii, multidrug resistant Pseudomonas aeruginosa, and carbapenem-resistant Enterobacteriaceae) pathogens to predict antimicrobial resistance or hypervirulence. Several biomarkers of drug-resistant genotypes in S. aureus, A. baumannii, P. aeruginosa, and K. pneumoniae, as well as hypervirulence in C. difficile, were detected. The fastest possible susceptibility testing with MALDI-TOF MS is simultaneous detection of a characteristic drug-resistant peak and species identification in the same spectra generated in routine processing. According to our approach, resistance or virulence biomarker peaks can be identified while performing routine microbiology analysis, and no additional assays nor prolonged incubation time is needed. Outstanding biomarker peaks detected in our study should be further analysed by additional methods to identify the specific proteins involved.

Clostridium difficile infection in patients with chronic kidney disease in Mexico
.

BACKGROUND: Clostridium difficile infection (CDI) is a leading cause of healthcare-associated diarrhea worldwide. Patients with chronic kidney disease (CKD) are especially vulnerable, as they are exposed to CDI risk factors including frequent antibiotics. MATERIALS AND METHODS: In order to identify the risk factors for CDI in CKD patients, a 33-month long case-control study was carried out at a tertiary-care hospital in Mexico. CDI was confirmed at the genetic level, and univariate and multivariate analyses were performed to identify the association between risk factors, biomarkers, and outcome options (survival, relapse, death). RESULTS: Among the 1,198 patients with healthcare-associated diarrhea, 354 (29.5%) were CDI cases. 105 (29.6%) CDI cases and 192 (22.7%) controls had CKD. 84 (80%) CKD+CDI cases had a favorable outcome, 10 (9.5%) relapsed, and the 3-month mortality rate included 11 (10.4%) patients. Compared with controls, CDI cases had more previous hospitalizations (63.8 vs. 46.9%, p = 0.005), abdominal distension (46.7 vs. 36.5%, p = 0.056), abdominal pain (60.0 vs. 41.1%, p = 0.002), and polymorphonuclear leukocyte in stools (71.4 vs. 40.5%, p = 0.001) as well as poorer outcomes at 3 months. The patients in the 027-strain group were older, and most of the patients had CKD stage 5 (88.5% vs. 71.1%, p = 0.007), while CKD stage-4 patients were more frequently infected with non-027 strains. In the multivariate analysis of risk factors for CDI, only previous antibiotic exposure (odds ratio = 2.01, 95% confidence interval: 1.05 - 3.84; p = 0.034) was independently associated with CDI in patients with CKD stage 5. CONCLUSION: Mexican patients with CKD are at risk for CDI. This susceptible group should be protected by promoting appropriate guidelines.
.

Circulation of Highly Drug-Resistant Clostridium difficile Ribotypes 027 and 001 in Two Tertiary-Care Hospitals in Mexico.

OBJECTIVE: To assess drug susceptibility and characterize Clostridium difficile ribotypes in isolates from two tertiary-care hospitals in Mexico. METHODS: Isolates were evaluated for genotyping, antimicrobial susceptibility testing and detection of mutations associated with drug resistance. PCR ribotyping was performed using a combination of gel-based and capillary electrophoresis-based approaches. RESULTS: MIC50 and MIC90 were ≥128 mg/L for ciprofloxacin, erythromycin, clindamycin, and rifampicin. There was no reduced susceptibility to metronidazole or tetracycline; however, reduced susceptibility to vancomycin (≥4 mg/L) and fidaxomicin (≥2 mg/L) was detected in 50 (40.3%) and 4 (3.2%) isolates, respectively. Furthermore, the rpoB Arg505Lys mutation was more frequently detected in isolates with high minimum inhibitory concentration (MIC) to rifampicin (≥32 mg/L) (OR = 52.5; 95% CI = 5.17-532.6; p < 0.000). Of the 124 C. difficile isolates recovered, 84 (66.7%) were of ribotype 027, 18 (14.5%) of ribotype 001, and the remainder were other ribotypes (353, 255, 220, 208, 176, 106, 076, 020, 019, 017, 014, 012, 003, and 002). CONCLUSION: Ribotypes 027 and 001 were the most frequent C. difficile isolates recovered in this study, and demonstrated higher MICs. Furthermore, we found four isolates with reduced susceptibility to fidaxomicin, raising a concern since this drug is currently unavailable in Mexican Hospitals.