Drug Resistance in Biofilm and Planktonic Cells of Achromobacter spp., Burkholderia spp., and Stenotrophomonas maltophilia Clinical Isolates.

Background: Biofilm production in nonfermenting Gram-negative bacteria influences drug resistance. The aim of this work was to evaluate the effect of different antibiotics on biofilm eradication of clinical isolates of Achromobacter, Burkholderia, and Stenotrophomonas maltophilia. Methods: Clinical isolates were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry in a third-level hospital in Monterrey, Mexico. Crystal violet staining was used to determine biofilm production. Drug susceptibility testing was determined by broth microdilution in planktonic cells and biofilm cells. Results: Resistance in planktonic cells was moderate to trimethoprim-sulfamethoxazole, and low to chloramphenicol, minocycline, levofloxacin (S. maltophilia and Burkholderia), ceftazidime, and meropenem (Burkholderia and Achromobacter). Biofilm eradication required higher drug concentrations of ceftazidime, chloramphenicol, levofloxacin, and trimethoprim-sulfamethoxazole than planktonic cells (p < 0.05). Levofloxacin showed biofilm eradication activity in S. maltophilia, minocycline and meropenem in Burkholderia, and meropenem in Achromobacter. Conclusions: Drug resistance increased due to biofilm production for some antibiotics, particularly ceftazidime and trimethoprim-sulfamethoxazole for all three pathogens, chloramphenicol for S. maltophilia and Burkholderia, and levofloxacin for Burkholderia. Some antibiotics could be used for the treatment of biofilm-associated infections in our population, such as levofloxacin for S. maltophilia, minocycline and meropenem for Burkholderia, and meropenem for Achromobacter.

Biofilm Eradication and Inhibition of Methicillin-resistant Staphylococcus Clinical Isolates by Curcumin-Chitosan Magnetic Nanoparticles.

Biofilm-producing methicillin-resistant Staphylococcus aureus (MRSA) and coagulase-negative Staphylococci (MR-CoNS) are a clinical challenge for the treatment of healthcare-associated infections. As alternative antimicrobial options are needed, we aimed to determine the effect of curcumin-chitosan magnetic nanoparticles on the biofilm of staphylococcal clinical isolates. MRSA and CoNS clinical isolates were identified by MALDI-TOF mass spectrometry. Antimicrobial susceptibility testing was performed by broth microdilution. Nanoparticles were synthesized by co-precipitation of magnetic nanoparticles (MNP) and encapsulation by ionotropic gelation of curcumin (Cur) and chitosan (Chi). Biofilm inhibition and eradication by nanoparticles with and without the addition of oxacillin was assessed on staphylococcal strains. Cur-Chi-MNP showed antimicrobial activity on planktonic cells of MRSA and MR-CoNS strains and inhibited biofilm of MRSA. The addition of OXA to Cur-Chi-MNP increased biofilm inhibition and eradication activity against all Staphylococci strains (p=0.0007); higher biofilm activity was observed in early biofilm stages. Cur-Chi-MNP showed antimicrobial and biofilm inhibition activity against S. aureus. The addition of OXA increased biofilm inhibition and eradication activity against all Staphylococci strains. A combination treatment of Cur-Chi-MNP and OXA could be potentially used to treat staphylococcal biofilm-associated infections in its early stages before the establishment of biofilm bacterial cells.

Antibacterial efficacy of novel bismuth-silver nanoparticles synthesis on Staphylococcus aureus and Escherichia coli infection models.

Introduction: The emergence of multi-drug-resistant bacteria is one of the main concerns in the health sector worldwide. The conventional strategies for treatment and prophylaxis against microbial infections include the use of antibiotics. However, these drugs are failing due to the increasing antimicrobial resistance. The unavailability of effective antibiotics highlights the need to discover effective alternatives to combat bacterial infections. One option is the use of metallic nanoparticles, which are toxic to some microorganisms due to their nanometric size. Methods: In this study we (1) synthesize and characterize bismuth and silver nanoparticles, (2) evaluate the antibacterial activity of NPs against Staphylococcus aureus and Escherichia coli in several infection models (in vivo models: infected wound and sepsis and in vitro model: mastitis), and we (3) determine the cytotoxic effect on several cell lines representative of the skin tissue. Results and discussion: We obtained bimetallic nanoparticles of bismuth and silver in a stable aqueous solution from a single reaction by chemical synthesis. These nanoparticles show antibacterial activity on S. aureus and E. coli in vitro without cytotoxic effects on fibroblast, endothelial vascular, and mammary epithelium cell lines. In an infected-wound mice model, antibacterial effect was observed, without effect on in vitro mastitis and sepsis models.

Genome Sequences of Bordetella pertussis Isolates from Outbreaks in Northeastern Mexico.

Here, we report the draft genome sequences of 4 Bordetella pertussis isolates which correspond to major clones isolated between 2008 and 2014 from two outbreaks in northeastern Mexico. The B. pertussis clinical isolates belong to the ptxP3 lineage, and they are grouped into two major clusters, defined by the fimH allele.

Antibiofilm and antimicrobial activity of curcumin-chitosan nanocomplexes and trimethoprim-sulfamethoxazole on Achromobacter, Burkholderia, and Stenotrophomonas isolates.

BACKGROUND: Non-fermenting Gram-negative Achromobacter xylosoxidans, Burkholderia cepacia complex, and Stenotrophomonas maltophilia species cause healthcare-associated infections, often showing resistance to first-line drugs such as trimethoprim-sulfamethoxazole (TMP-SXT). The aim of this study was to determine the effect of curcumin-chitosan nanocomplexes on biofilm-producing clinical isolates of non-fermenting Gram-negative bacilli. METHODS: A. xylosoxidans, B. cepacia complex, and S. maltophilia clinical isolates were identified by MALDI-TOF mass spectrometry. Antimicrobial susceptibility was determined by broth microdilution. Curcumin (Cur), chitosan (Chi), and sodium tripolyphosphate (TPP) were encapsulated by ionotropic gelation in magnetic nanoparticles (MNP) and were assessed by scanning electron microscopy (SEM) and Fourier-transform infrared (FTIR). Biofilm inhibition and eradication by Cur-Chi-TPP-MNP with TMP-SXT was assessed. RESULTS: Cur-Chi-TPP-MNP in combination with TMP-SXT showed biofilm inhibition activity in A. xylosoxidans (37.5 µg/mL), B. cepacia (18.75 µg/mL), and S. maltophilia (4.69-18.75 µg/mL) and low biofilm eradication activity in all three strains (150 - 300 µg/mL). CONCLUSIONS: Cur-Chi-TPP-MNP in combination with TMP-SXT was able to inhibit biofilm and in lower effect to eradicate established biofilms of clinical isolates of A. xylosoxidans, B. cepacia complex, and S. maltophilia species. Our results highlight the need to assess these potential treatment options to be used clinically in biofilm-associated infections.

Rapid methicillin resistance detection and subspecies discrimination in Staphylococcus hominis clinical isolates by MALDI-TOF MS.

PURPOSE: Staphylococcus hominis is a coagulase-negative opportunistic pathogen responsible for implanted medical device infections. Rapid identification and virulence factors detection are crucial for appropriate antimicrobial therapy. We aimed to search protein biomarker peaks for rapid classification of antibiotic resistance and subspecies of S. hominis using MALDI-TOF MS. METHODS: S. hominis clinical isolates (n = 148) were screened for subspecies differentiation by novobiocin resistance. Biofilm composition and formation were determined by detachment assay and crystal violet staining, respectively. Antibiotic susceptibility was performed by the broth microdilution method. The search for potential biomarkers peaks was enabled by ClinProTools 3.0, flexAnalysis 3.4, and Biotools 3.2 for statistical analysis, peak visualization, and protein/peptide alignment, respectively. RESULTS: Of 148 isolates, 12.16% were classified as S. hominis subsp. novobiosepticus, 77.77% were biofilm producers, and ˃ 50% were multidrug-resistant. Two potential biomarker peaks, 8975 m/z and 9035 m/z were detected for the discrimination of methicillin resistance with a sensitivity of 96.72%. The following peaks were detected for subspecies differentiation: 2582 m/z, 2823 m/z, and 2619 m/z with 88.89-98.28% of sensitivity. CONCLUSIONS: We found potential biomarker peaks to predict methicillin resistance and discriminate S. hominis subspecies during routine MALDI-TOF MS identification in a clinical setting to enable better antibiotic treatment.

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.

Improvement of antimicrobial susceptibility testing in biofilm-growingcoagulase-negative Staphylococcus hominis.

Coagulase-negative Staphylococcus hominis causes bloodstream infections and often can form biofilms on medical devices. This study aimed to improve the current methodology for antimicrobial susceptibility testing (AST) in biofilm-growing S. hominis isolates. Biofilm production of S. hominis was assessed using the crystal violet staining method in trypticase soy broth supplemented with 1% glucose (TSBglu1%), Mueller-Hinton broth (MHB), or MHBglu1% using flat-bottom plates or the Calgary device. Susceptibility to antibiotics was assessed using the broth microdilution method (MHB and TSBglu1%) in planktonic cells (round-bottom plates) and biofilm cells (flat-bottom plates and the Calgary device). Biofilm determination using TSBglu1% yielded better performance over MHB, and flat-bottom plates without agitation were preferred over the Calgary device. Higher fold dilution values between the minimum biofilm eradication concentration (MBEC) and the minimum inhibitory concentration (MIC) were obtained in MHB for almost all antibiotics, except for linezolid. TSBglu1% and flat-bottom polystyrene plates were preferred over MHB and the Calgary device for biofilm determination. AST in biofilm-growing S. hominis showed better performance using TSBglu1% compared to MHB. Therefore, when comparing MBEC and MIC values, AST in planktonic cells could also be performed using TSBglu1% instead of MHB.

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.

Two New Dihydrosphingosine Analogs Against Mycobacterium tuberculosis Affect gltA1, lprQ, and rpsO Expression.

The emergence of multidrug-resistant (MDR) Mycobacterium tuberculosis strains threaten the control of tuberculosis. New antitubercular dihydrosphingosine analogs, named UCIs, have been evaluated in preclinical studies but their cellular and molecular mechanisms of action against M. tuberculosis are still unknown. The aim of this study was to evaluate the effect of UCI exposure on gene expression of drug-sensitive H37Rv and MDR CIBIN:UMF:15:99 clones of M. tuberculosis which were isolated, phenotypically, and genetically characterized, cultured to log phase and treated with UCI compounds; followed by total RNA isolation, reverse transcription and hybridization assays on Affymetrix genomic microarrays. Data were validated with RT-qPCR assays. As results, UCI-05 and UCI-14 exposure increased gltA1 expression in drug-sensitive H37Rv clones. Furthermore, UCI-05 increased lprQ expression in MDR CIBIN:UMF:15:99 M. tuberculosis clones while UCI-14 reduced the expression of this gene in drug-sensitive H37Rv clones. In addition, UCI-05 reduced rpsO expression in drug-sensitive H37Rv clones. We found gene expression alterations that suggest these molecules may alter carbon and lipid metabolism as well as interfere in the protein-producing machinery in M. tuberculosis.

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.

Discrimination of biofilm-producing Stenotrophomonas maltophilia clinical strains by matrix-assisted laser desorption ionization-time of flight.

Stenotrophomonas maltophilia is a Gram-negative drug-resistant pathogen responsible for healthcare-associated infections. The aim was to search for biomarker peaks that could rapidly detect biofilm production in S. maltophilia clinical isolates obtained from two tertiary care hospitals in Mexico. Isolates were screened for the presence of biofilm-associated genes, in which the fsnR gene was associated with biofilm production (p = 0.047), whereas the rmlA+ genotype was associated with the rpfF- genotype (p = 0.017). Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectra comparison yielded three potential biomarker peaks (4661, 6074, and 6102 m/z) of biofilm-producing rmlA+ and rpfF- genotypes with >90% sensitivity (p<0.001). MALDI-TOF MS analyses showed a correlation between the relative abundance of 50S ribosomal proteins (L30 and L33) and the presence of the fnsR, rmlA and rpfF-2 genes, suggested to play a role in biofilm formation. Isolates obtained in the intensive care unit showed low clonality, suggesting no transmission within the hospital ward. The detection of biomarkers peaks by MALDI-TOF MS could potentially be used to early recognize and discriminate biofilm-producing S. maltophilia strains and aid in establishing appropriate antibiotic therapy.

Acquired Genetic Elements that Contribute to Antimicrobial Resistance in Frequent Gram-Negative Causative Agents of Healthcare-Associated Infections.

Antimicrobial resistance (AMR) is a worldwide public health problem that reduces therapeutic options and increases the risk of death. The causative agents of healthcare-associated infections (HAIs) are drug-resistant microorganisms of the nosocomial environment, which have developed different mechanisms of AMR. The hospital-associated microbiota has been proposed to be a reservoir of genes associated with AMR and an environment where the transfer of genetic material among organisms may occur. The ESKAPE group (Enterococcus faecalis and Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter aerogenes and Escherichia coli) is a frequent causative agents of HAIs. In this review, we address the issue of acquired genetic elements that contribute to AMR in the most frequent Gram-negative of ESKAPE, with a focus on last resort antimicrobial agents and the role of transference of genetic elements for the development of AMR.

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.