Essential Oil of Fractionated Oregano as Motility Inhibitor of Bacteria Associated with Urinary Tract Infections.

In this research, several analyses were carried out on concentrated fractions of Mexican oregano essential oil (Poliomintha longiflora Gray) in order to determine its ability to inhibit the growth and the motility of Escherichia coli (swimming), Pseudomonas aeruginosa (swimming), and Proteus vulgaris (swarming); these Gram-negative bacteria associated with urinary tract infections are motile due to the presence of flagella, which is considered an important virulence factor that favors their motility when trying to reach the target organ and cause an infection. Also, the resistance pattern to antibiotics of each strain was determined. The results showed resistance pattern (8 out of 12 antibiotics tested) for P. aureginosa, while E. coli and P. vulgaris were resistant to 4 antibiotics out of the 12 tested. On the other hand, fractionated oregano caused an inhibition of growth and a reduction in motility, varying between fractions and among bacteria. Fraction 4 showed major growth reduction, with MBC values ranging from 0.002 to 23.7 mg/mL. Treatment with fractionated oregano (F1, F2, F3, F4) reduced the motility by 92-81% for P. vulgaris, 90-83% for E. coli, and 100-8.9% for P. aeruginosa. These results demonstrated a higher performance with a lower application dose due to its high content of Carvacrol and Thymol; unlike other concentrated fractions, this synergy of oxygenated monoterpenes may cause greater antimicrobial activity.

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) pose clinical challenges in treating healthcare-associated infections. As alternative antimicrobial options are needed, in this study, we aimed to determine the effect of curcumin-chitosan magnetic nanoparticles (Cur-Chi-MNP) on the biofilms of staphylococcal clinical isolates. MRSA and CoNS clinical isolates were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Antimicrobial susceptibility testing was performed using the broth microdilutions. Nanoparticles were synthesized by the co-precipitation of magnetic nanoparticles (MNP) and encapsulated by the ionotropic gelation of curcumin (Cur) and chitosan (Chi). Biofilm inhibition and eradication by nanoparticles, with and without the addition of oxacillin (OXA), were assessed in Staphylococcus strains. Cur-Chi-MNP showed antimicrobial activity against planktonic cells of MRSA and MR-CoNS strains and inhibited MRSA biofilm. The addition of OXA to Cur-Chi-MNP increased the biofilm inhibition and eradication activity against all staphylococcal strains (P = 0.0007), and higher biofilm activity was observed in the early biofilm stages. Cur-Chi-MNP showed antimicrobial and biofilm inhibitory activities against S. aureus. Addition of OXA increased biofilm inhibition and eradication activity against all staphylococcal strains. A combination treatment of Cur-Chi-MNP and OXA could potentially be used to treat staphylococcal biofilm-associated infections in the 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.

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.

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.

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.

High sporulation and overexpression of virulence factors in biofilms and reduced susceptibility to vancomycin and linezolid in recurrent Clostridium [Clostridioides] difficile infection isolates.

Clostridium [Clostridioides] difficile infection (CDI) is one of the leading causes of diarrhea associated with medical care worldwide, and up to 60% of patients with CDI can develop a recurrent infection (R-CDI). A multi-species microbiota biofilm model of C. difficile was designed to evaluate the differences in the production of biofilms, sporulation, susceptibility to drugs, expression of sporulating (sigH, spo0A), quorum sensing (agrD1, and luxS), and adhesion-associated (slpA and cwp84) pathway genes between selected C. difficile isolates from R-CDI and non-recurrent patients (NR-CDI). We obtained 102 C. difficile isolates from 254 patients with confirmed CDI (66 from NR-CDI and 36 from R-CDI). Most of the isolates were biofilm producers, and most of the strains were ribotype 027 (81.374%, 83/102). Most C. difficile isolates were producers of biofilm (100/102), and most were strongly adherent. Sporulation was higher in the R-CDI than in the NR-CDI isolates (p = 0.015). The isolates from R-CDI patients more frequently demonstrated reduced susceptibility to vancomycin than isolates of NR-CDI patients (27.78% [10/36] and 9.09% [6/66], respectively, p = 0.013). The minimum inhibitory concentrations for vancomycin and linezolid against biofilms (BMIC) were up to 100 times and 20 times higher, respectively, than the corresponding planktonic MICs. Expression of sigH, spo0A, cwp84, and agrD1 was higher in R-CDI than in NR-CDI isolates. Most of the C. difficile isolates were producers of biofilms with no correlation with the ribotype. Sporulation was greater in R-CDI than in NR-CDI isolates in the biofilm model of C. difficile. The R-CDI isolates more frequently demonstrated reduced susceptibility to vancomycin and linezolid than the NR-CDI isolates in both planktonic cells and biofilm isolates. A higher expression of sporulating pathway (sigH, spo0A), quorum sensing (agrD1), and adhesion-associated (cwp84) genes was found in R-CDI than in NR-CDI isolates. All of these factors can have effect on the recurrence of the infection.

Virulence Factors of Clostridioides (Clostridium) difficile Linked to Recurrent Infections.

From 20 to 30% of Clostridioides (Clostridium) difficile infection (CDI), patients might develop recurrence of the infection (RCDI) and, after the first recurrence, the risk of further episodes increases up to 60%. Several bacterial virulence factors have been associated with RCDI, including the elevated production of toxins A and B, the presence of a binary toxin CDT, and mutations in the negative regulator of toxin expression, tcdC. Additional factors have shown to regulate toxin production and virulence in C. difficile in RCDI, including the accessory-gene regulator agr, which acts as a positive switch for toxin transcription. Furthermore, adhesion and motility-associated factors, such as Cwp84, SlpA, and flagella, have shown to increase the adhesion efficiency to host epithelia, cell internalization, and the formation of biofilm. Finally, biofilm confers to C. difficile protection from antibiotics and acts as a reservoir for spores that allow the persistence of the infection in the host. In this review, we describe the key virulence factors of C. difficile that have been associated with recurrent infections.

Mycobacterium tuberculosis promotes genomic instability in macrophages

BACKGROUND Mycobacterium tuberculosis is an intracellular pathogen, which may either block cellular defensive mechanisms and survive inside the host cell or induce cell death. Several studies are still exploring the mechanisms involved in these processes. OBJECTIVES To evaluate the genomic instability of M. tuberculosis-infected macrophages and compare it with that of uninfected macrophages. METHODS We analysed the possible variations in the genomic instability of Mycobacterium-infected macrophages using the DNA breakage detection fluorescence in situ hybridisation (DBD-FISH) technique with a whole human genome DNA probe. FINDINGS Quantitative image analyses showed a significant increase in DNA damage in infected macrophages as compared with uninfected cells. DNA breaks were localised in nuclear membrane blebs, as confirmed with DNA fragmentation assay. Furthermore, a significant increase in micronuclei and nuclear abnormalities were observed in infected macrophages versus uninfected cells. MAIN CONCLUSIONS Genomic instability occurs during mycobacterial infection and these data may be seminal for future research on host cell DNA damage in M. tuberculosis infection.

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.