Vancomycin Resistance Research Articles

Antibiotic Susceptibility of Staphylococcus Aureus with VanA and MecA Genes.

Staphylococcus aureus (S.aureus) is an emerging antibiotic resistant bacterium responsible for various infections in human. Resistance to methicillin and vancomycin are of prime concern in S. aureus. The study aims to determine the minimum inhibitory concentration (MIC) of Vancomycin and evaluate the existence of mecA and vanA genes, associated with antibiotic resistance. Clinical specimens from three Kathmandu hospitals were processed and S. aureus was identified using conventional microbiological procedures. MRSA was phenotypically identified with cefoxitin (30µg) disc diffusion, while vancomycin susceptibility was assessed using the Ezy MICTM stripes. The mecA and vanA genes were detected by polymerase chain reaction (PCR). Out of 266 S. aureus samples from various clinical specimen subjected for analysis, 77 (28.9%) were found methicillin-resistant (MRSA) and 10 (3.8%) were observed vancomycin-resistant (VRSA). Vancomycin resistant isolates showed a significant correlation between resistance to ampicillin, chloramphenicol, and cefoxitin. The mecA gene was found in 39 of the MRSA isolates, having 50.64% of MRSA cases, while the vanA gene was detected in 4 of the VRSA cases, constituting 40% of VRSA occurrences. The strains with higher vancomycin minimum inhibitory concentration values (≥ 1.5 μg/ml) displayed increased resistance rates to various antibiotics compared to strains with lower minimum inhibitory concentration values (< 1.5 μg/ml). The presence of vanA genes was strongly associated (100%) with vancomycin resistance, while the 10.3% mecA gene was identified from MRSA having resistance towards vancomycin also.

Detection of Vancomycin Resistance among Methicillin-Resistant Staphylococcus aureus Strains Recovered from Children with Invasive Diseases in a Reference Pediatric Hospital.

Vancomycin is the cornerstone in treating methicillin-resistant Staphylococcus aureus (MRSA) infections. However, therapeutic failures can occur when MRSA strains with decreased susceptibility to glycopeptides (DSG) are involved. The aim of this study was to detect and characterize DSG in MRSA recovered from children with invasive diseases at a reference pediatric hospital between 2009 and 2019. Fifty-two MRSA strains were screened using agar plates with vancomycin 3 and 4 mg/L (BHI-3 and BHI-4); the VITEK2 system; and standard and macro E-tests. Suspicious hVISA were studied by population analysis profiling-area under the curve (PAP-AUC), and wall thickness was analyzed by transmission electron microscopy. Neither VRSA nor VISA were detected in this set. As only three strains met the hVISA criteria, the PAP-AUC study included 12 additional MRSA strains that grew one colony on BHI-4 plates or showed minimum inhibitory concentrations of vancomycin and/or teicoplanin ≥ 1.5 mg/L. One strain was confirmed as hVISA by PAP-AUC. The wall thickness was greater than the vancomycin-susceptible control strain; it belonged to ST30 and carried SCCmec IV. As expected, a low frequency of hVISA was found (1.9%). The only hVISA confirmed by PAP-AUC was not detected by the screening methods, highlighting the challenge that its detection represents for microbiology laboratories.

Metabolic Profiling Reveals the Importance of Arginine Metabolism via the Arginine Deiminase Pathway in Vancomycin-intermediate S. aureus

Gene mutations located in VraSR and GraSR, two-component regulatory systems have been reported to cause vancomycin resistance in vancomycin-intermediate Staphylococcus aureus (VISA). Subsequent comparative proteomic profiling between vancomycin-susceptible S. aureus (VSSA) and VISAs of the Mu50 genetic lineage revealed up-regulated level of catabolic ornithine carbamoyltransferase (ArcB) in the latter, suggesting a role of arginine catabolism in VISA development. This study aimed to further investigate metabolic pathways associated with VISA development using liquid chromatography mass spectrometry (LCMS)-based untargeted metabolomic profiling. Metabolite profiles were compared among 3 isogenic strains of the Mu50 lineage: Mu50Ω (VSSA), Mu50Ω-vraSm (VISA) and Mu50Ω-vraSm-graRm (VISA). Intracellular levels of α-hydroxyglutaric acid, choline, lysine, malic acid, N-acetylornithine, nicotinamide, and cystathionine were found to be significantly different between VISAs compared to VSSA. VISA cells were found to have lower levels of intracellular citrulline and extracellular arginine, with higher extracellular level of ornithine compared to VSSA. These differences in metabolite levels between VISA and VSSA suggested the importance of arginine metabolism. Activation of the arginine deiminase pathway (ADI) in VISAs of the Mu50 lineage could be further explored as a target for antibiotic development.

Identification of vanA gene on Vancomycin-Resistant Staphylococcus aureus from Diabetic Ulcer Isolate at Lampung Province

Staphylococcus aureus is a type of bacteria that causes an increasing number of infections in hospitals, particularly in diabetic ulcers. Over the last few decades, there has been an alarming increase in the prevalence of pathogen strains of Staphylococcus aureus resistant to antibiotics such as Vancomycin. This study aimed to identify the presence of the vanA gene, which is responsible for the mode of Vancomycin resistance in Staphylococcus aureus Lampung isolate. Ulcer swab was collected from 32 patients with ulcer complications from surgical Installation, Internal Medicine, Home Diabetes Wound Clinic and Diabetes Wound Center in Bandar Lampung. Among the total ulcer swab, 12 samples of S. aureus were identified and subjected to the Minimum Inhibitory Concentration test to identify Vancomycin Resistant Staphylococcus aureus and evaluated the vanA gene by Polymerase Chain Reaction. To detect the presence of the vanA gene, a Polymerase Chain Reaction was performed on Staphylococcus aureus, Vancomycin-Resistant Staphylococcus aureus, and Vancomycin-intermediate Staphylococcus aureus using a specific primer arrangement. There were two samples of Vancomycin- Resistant Staphylococcus aureus and one sample of Vancomycin-intermediate Staphylococcus aureus from the Minimum Inhibitory Concentration test but only one sample tested positive for the vanA gene on Polymerase Chain Reaction. There is Staphylococcus aureus resistance to Vancomycin in Lampung isolate and the vanA gene was detected in some resistant isolates. The arrangement of the vanA gene in the Lampung isolate is different from that of the vanA gene in other places isolates.

Prevalence and Molecular Characterization of Vancomycin Variable Enterococcus faecium Isolated From Clinical Specimens.

Vancomycin variable Enterococcus (VVE) bacteria are phenotypically susceptible to vancomycin, but they harbor the vanA gene. We aimed to ascertain the prevalence of VVE among clinically isolated vancomycin-susceptible Enterococcus faecium (VSE) isolates, as well as elucidate the molecular characteristics of the vanA gene cluster within these isolates. Notably, we investigated the prevalence and structure of the vanA gene cluster of VVE. Between June 2021 and May 2022, we collected consecutive, non-duplicated vancomycin-susceptible Enterococcus faecium (VSE) samples. Real-time PCR was performed to detect the presence of vanA, vanB, and vanC. Overlapping PCR with sequencing and whole-genome sequencing were performed for structural analysis. Sequence types (STs) were determined by multilocus sequence typing. Exposure testing was performed to assess the ability of the isolates to acquire vancomycin resistance. Among 282 VSE isolates tested, 20 isolates (7.1%) were VVE. Among them, 17 isolates had partial deletions in the IS1216 or IS1542 sequences in vanS (N=10), vanR (N=5), or vanH (N=2). All VVE isolates belonged to the CC17 complex and comprised five STs, namely ST17 (40.0%), ST1421 (25.0%), ST80 (25.0%), ST787 (5.0%), and ST981 (5.0%). Most isolates were related to three hospital-associated clones (ST17, ST1421, and ST80). After vancomycin exposure, 18 of the 20 VVEs acquired vancomycin resistance. Considering the high reversion rate, detecting VVE by screening VSE for vanA is critical for appropriate treatment and infection control.

Genome mining of Escherichia coli WG5D from drinking water source: unraveling antibiotic resistance genes, virulence factors, and pathogenicity

BackgroundEscherichia coli, a ubiquitous inhabitant of the gut microbiota, has been recognized as an indicator of fecal contamination and a potential reservoir for antibiotic resistance genes. Its prevalence in drinking water sources raises concerns about the potential dissemination of antibiotic resistance within aquatic ecosystems and the subsequent impact on public health. The ability of E. coli to acquire and transfer resistance genes, coupled with the constant exposure to low levels of antibiotics in the environment, underscores the need for comprehensive surveillance and rigorous antimicrobial stewardship strategies to safeguard the quality and safety of drinking water supplies, ultimately mitigating the escalation of antibiotic resistance and its implications for human well-being.MethodsWG5D strain, isolated from a drinking water distribution source in North-West Province, South Africa, underwent genomic analysis following isolation on nutrient agar, anaerobic cultivation, and DNA extraction. Paired-end Illumina sequencing with a Nextera XT Library Preparation kit was performed. The assembly, annotation, and subsequent genomic analyses, including phylogenetic analysis using TYGS, pairwise comparisons, and determination of genes related to antimicrobial resistance and virulence, were carried out following standard protocols and tools, ensuring comprehensive insights into the strain’s genomic features.ResultsThis study explores the notable characteristics of E. coli strain WG5D. This strain stands out because it possesses multiple antibiotic resistance genes, encompassing tetracycline, cephalosporin, vancomycin, and aminoglycoside resistances. Additionally, virulence-associated genes indicate potential heightened pathogenicity, complemented by the identification of mobile genetic elements that underscore its adaptability. The intriguing possibility of bacteriophage involvement and factors contributing to pathogenicity further enriches our understanding. We identified E. coli WG5D as a potential human pathogen associated with a drinking water source in South Africa. The analysis provided several antibiotic resistance-associated genes/mutations and mobile genetic elements. It further identified WG5D as a potential human pathogen. The occurrence of E. coli WG5D raised the awareness of the potential pathogens and the carrying of antibiotic resistance in drinking water.ConclusionsThe findings of this study have highlighted the advantages of the genomic approach in identifying the bacterial species and antibiotic resistance genes of E. coli and its potential as a human pathogen.

COMPARISON OF NASAL CARRIAGE OF MRSA AND ITS ANTIBIOTIC SUSCEPTIBILITY WITH SPECIAL REFERENCE TO MUPIROCIN AMONG STUDENTS OF HEALTH-CARE SETTINGS AND OTHER STUDENTS

Objectives: One of the potential risk factors for nosocomial staphylococcal and Methicillin-resistant Staphylococcus aureus (MRSA) infections is colonization of the anterior nares of HCWs, and from there they may spread to the patients, which may cause a burden on the health-care system. In this context, we made an attempt to compare the Staphylococcal and MRSA nasal carriage and their susceptibility to Mupirocin in students who are exposed to the health-care system and those who are not. Methods: Nasal swabs were collected from 100 paramedical students as the study group and 100 non-medical students as the control group, all in the age group between 18 and 21 years. 5% sheep blood agar and mannitol salt agar were used to isolate S. aureus, and antibiotic sensitivity was done by Kirby Bauer Disc Diffusion Technique. Cefoxitin disc (30 μg) and Vancomycin screen agar were used to detect MRSA and Vancomycin resistance, respectively. A 5 μg disc of Mupirocin was used to test the susceptibility of Mupirocin. Results: Nasal carriage rate of S. aureus and MRSA was 23% and 8% in Paramedical students and 17% and 4% in Nonmedical students respectively. 83% of isolates from paraclinical students and 95% of isolates from non-medical students were susceptible to linezolid. Out of 12 MRSA strains from both groups only one strain showed resistance to Mupirocin. Conclusions: Nasal carriage of S. aureus and MRSA and antibiotic resistance of isolated strains were more common in paramedical students and also in female students. Vancomycin resistance was not observed in MRSA strains from male students. Mupirocin resistance, even in a single case, needs to be addressed.

Polycystic intrahepatic infection caused by Enterococcus casseliflavus: a case report and literature review

BackgroundEnterococcus casseliflavus is a rare pathogenic bacterium that is characterized by vancomycin resistance and can lead to multiple infections in the human body. This report describes a rare case of polycystic intrahepatic infection with E. casseliflavus which necessitated antibiotic treatment and surgical intervention involving cystic drainage.Case PresentationA 59-year-old woman, a long-term hemodialysis patient, was hospitalized due to a 5-day history of fever, abdominal pain, and diarrhea, which were possibly caused by the ingestion of contaminated food. Her blood culture yielded a positive result for E. casseliflavus, and she was initially treated with piperacillin/tazobactam and linezolid. Later, the antibiotic regimen was adjusted to include meropenem and linezolid. Despite treatment, her body temperature remained elevated. However, subsequent blood cultures were negative for E.casseliflavus.Conventional CT scans and ultrasound examinations did not identify the source of infection. However, a PET-CT examination indicated an intrahepatic cyst infection. Following MRI and ultrasound localization, percutaneous intrahepatic puncture and drainage were performed on the 20th day. Fluoroquinolones were administered for 48 days. On the 32nd day, MRI revealed a separation within the infected cyst, leading to a repeat percutaneous drainage at a different site. Subsequently, the patient’s temperature returned to normal. The infection was considered resolved, and she was discharged on the 62nd day. Follow-up results have been favorable thus far.ConclusionsBased on the findings from this case, it is recommended to promptly conduct PET-CT examination to exclude the possibility of intracystic infection in cases of polycystic liver infection that are challenging to control. Furthermore, timely consideration should be given to puncture drainage in difficult cases.

Timing and clinical risk factors for early acquisition of gut pathogen colonization with multidrug resistant organisms in the intensive care unit

BackgroundMicrobiome restitution therapies are being developed to prevent gut pathogen colonization among patients in the intensive care unit (ICU) and in other select populations. If preventive therapies are to be effective, they must be administered prior to pathogen acquisition. The timing and risk factors for early acquisition of gut pathogen colonization (within 72 h) are currently unknown and could be helpful to guide ICU trial design.MethodsThis was a prospective cohort study. Patients in the ICU had deep rectal swabs performed within 4 h of ICU admission and exactly 72 h later. Early gut pathogen colonization was classified as the new presence (based on culture of rectal swabs) of one or more of the following organisms of interest: methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant (VRE), and Gram-negative bacteria that showed multidrug resistance (MDR) or third generation Cephalosporin resistance (Ceph-R). Clinical risk factors for early acquisition of gut pathogen colonization were captured using the Acute Physiology and Chronic Health Evaluation IV (APACHE IV) scoring system.FindingsAmong 131 patients who were swabbed at ICU admission and 72 h later, the rates of gut pathogen colonization at ICU admission were 11.4%, 10.6%, 38.6%, and 8.3% for MRSA, VRE, MDR and Ceph-R Gram-negatives respectively. Among the patients who were negative for a given pathogen at ICU admission, the rates of early acquisition of gut pathogen colonization were 7.8% for MRSA (95% CI 3.6 to 14.2%), 7.7% for VRE (95% CI 3.6 to 14.1%), 11.3% for MDR Gram-negatives (95% CI 4.4 to 18.8%), and 4.2% for Ceph-R Gram-negatives (95% CI 1.4 to 9.5%). There were no clinical risk factors which independently predicted early acquisition of gut pathogen colonization.InterpretationEarly gut pathogen colonization was common in the ICU, but our single-center study could not identify any clinical risk factors which were significantly associated with acquisition of gut pathogens.

Reduced Vancomycin Susceptibility in Clostridioides difficile Is Associated With Lower Rates of Initial Cure and Sustained Clinical Response.

Epidemiologic studies have shown decreasing vancomycin susceptibility among clinical Clostridioides difficile isolates, but the impact on patient outcomes is unknown. We hypothesized that reduced vancomycin susceptibility would be associated with decreased rates of sustained clinical response (SCR). This multicenter cohort study included adults with C. difficile infection (CDI) treated with oral vancomycin between 2016 and 2021. Clostridioides difficile isolates underwent agar dilution vancomycin susceptibility testing, ribotyping, and Sanger sequencing of the vancomycin resistance vanR gene. Reduced susceptibility was defined as vancomycin minimum inhibitory concentration (MIC) >2 μg/mL. The primary outcome was 30-day SCR; secondary outcomes were 14-day initial cure, 30-day recurrence, and 30-day mortality. Exploratory analysis assessed the association between the VanR Thr115Ala polymorphism, susceptibility, and outcomes. A high proportion (34% [102/300]) of C. difficile isolates exhibited reduced vancomycin susceptibility (range, 0.5-16 μg/mL; MIC50/90 = 2/4 μg/mL). Ribotype 027 accounted for the highest proportion (77.4% [41/53]) of isolates with reduced vancomycin susceptibility. Overall, 83% (249) of patients achieved 30-day SCR. Reduced vancomycin susceptibility was associated with lower rates of 30-day SCR (76% [78/102]) than vancomycin-susceptible strains (86% [171/198]; P = .031). A significantly lower rate of 14-day initial cure was also observed among individuals infected with strains with reduced vancomycin susceptibility (89% vs 96%; P = .04). Reduced susceptibility remained an independent predictor of 30-day SCR in multivariable modeling (odds ratio, 0.52 [95% confidence interval, .28-.97]; P = .04). Reduced vancomycin susceptibility in C. difficile was associated with decreased odds of 30-day SCR and lower 14-day initial cure rates in the studied patient cohort.

Risk factors and outcome associated with the acquisition of MDR linezolid-resistant Enterococcus faecium: a report from tertiary care centre.

The aim of the study was to determine the resistance profile of linezolid-resistant Enterococcus faecium (LREfm) and to investigate risk factors and outcomes associated with LREfm infections. A prospective case-control study was undertaken (2019 to 2022) and included 202 patients with LREfm infections (cases) and 200 controls with LSEfm infections. Clinical data was prospectively collected and analysed for risk factors and outcomes. Antimicrobial susceptibility was performed, and resistance profile was studied using WHOnet. Risk factors associated with LREfm infection were site of infection UTI (OR 5.87, 95% CI 2.59-13.29, p ≤ 0.001), prior use of carbapenem (OR 2.85 95% CI 1.62-5.02, p ≤ 0.001) and linezolid (OR 10.13, 95% CI 4.13-24.82, p ≤ 0.001), use of central line (OR 5.54, 95% CI 2.35-13.09, p ≤ 0.001), urinary catheter (OR 0.29, 95% CI 0.12-0.70, p ≤ 0.001) and ventilation (OR 14.87, 95% CI 7.86-28.11, p ≤ 0.007). The hospital stay 8-14 days (< 0.001) prior to infection and the mortality rate (p = 0.003) were also significantly high among patients with LREfm infections. Linezolid and vancomycin resistance coexisted; further, MDR, XDR and PDR phenotypes were significantly higher among LREfm. This study provided insight into epidemiology of MDR LREfm in a setting where linezolid use is high. The main drivers of infections with LREfm are multiple, including use of carbapenems and linezolid. Invasive procedures and increased hospital stay facilitate spread through breach in infection control practises. As therapeutic options are limited, ongoing surveillance of LREfm and VRE is critical to guide appropriate use of linezolid and infection control policies.

Bacteriophage and antibiotic combination therapy for recurrent Enterococcus faecium bacteremia.

Phage therapy is an emerging therapeutic approach for treating bacterial infections that do not respond to traditional antibiotics. The addition of phage therapy to systemic antibiotics to treat a patient with recurrent E. faecium infections that were non-responsive to antibiotics alone resulted in fewer hospitalizations and improved the patient's quality of life. Combination phage and antibiotic therapy reduced E. faecium and VRE abundance in the patient's stool. Eventually, an anti-phage antibody response emerged that was able to neutralize phage activity, which may have limited clinical efficacy. This study demonstrates the potential of phages as an additional option in the antimicrobial toolbox for treating invasive enterococcal infections and highlights the need for further investigation to ensure phage therapy can be deployed for maximum clinical benefit.

Antimicrobial resistance profile of Enterococcus species and molecular characterization of Vancomycin resistant Enterococcus faecium from the fecal samples of newly diagnosed adult HIV patients in Dar es Salaam, Tanzania

BackgroundEnterococci are becoming clinically more important especially among immunocompromised patients. Of concern are vancomycin resistant enterococci (VRE) which have both intrinsic and acquired forms of resistance. This work aimed to determine the antimicrobial resistance patterns of Enterococcus spp. and characterize VRE isolate obtained from HIV-infected patient using whole genome sequencing (WGS).Methods:Antimicrobial susceptibility testing was done on 57 enterococci isolates by both the disk diffusion method and Epsilometer test (E-Test). WGS was performed on VRE isolate determined by E-test.ResultsOut of the 57 enterococci isolates; 58% (33/57) were E. faecalis, 39% (22/57) E. faecium and 4% (2/57) were E. gallinarum. The highest antimicrobial resistance was observed in E. faecalis isolates. The most prevalent antimicrobial resistance was observed towards quinupristin-dalfopristin (56%, 32/57), followed by ciprofloxacin (28%), tigecycline (18%), daptomycin (16%), chloramphenicol (14%), ampicillin and teicoplanin (2%). Multidrug resistance (MDR) was detected in 11% (6/57) of the isolates. Vancomycin resistance and high-level gentamycin resistance (HLGR) were observed in one E. faecium and one E. faecalis isolates respectively. The VRE was typed as ST80, carried vanA and other resistance genes for aminoglycosides, tetracyclines, quinolones and ampicillin. Furthermore, the isolate had chromosomal mutations responsible for quinolone (gyrA (p.S83I) and parC (p.S80I) and ampicillin (pbp5) resistance.ConclusionsThe detection of VRE, HLGR and MDR in the study settings underscores the sustained surveillance of VRE in high-risk groups and institution of infection control measures for prompt identification and isolation of carriers to prevent the spread of VRE in the community and hospital settings.

Antimicrobial Resistance and Antibiotic Consumption in a Secondary Care Hospital in Mexico.

Antimicrobial resistance is a global health problem, due to morbidity, mortality, and healthcare costs. The misuse of antimicrobials is the main cause of antimicrobial resistance. The aim of this study was to report antimicrobial resistance and antibiotic consumption in a secondary care hospital in Mexico. Within a cross-sectional study, antimicrobial resistance data on ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) and antibiotic consumption from 2020 to 2022 were collected. Antimicrobial resistance was reported based on percentages of resistance and consumption was analyzed using the defined daily dose (DDD)/100 bed days and the AWaRe (Access, Surveillance, Reservation) antibiotic group. Antibiotic consumption in 2020, 2021 and 2022 was 330, 175 and 175 DDD/100 beds day, respectively. The rate of ceftriaxone resistance in E. coli (n = 526) and K. pneumoniae (n = 80) was 76% and 69%, respectively, the rate of carbapenem resistance in A. baumannii (n = 168) and P. aeruginosa (n = 108) was 92% and 52%, respectively; the rate of oxacillin resistance in S. aureus (n = 208) was 27%; and the rate of vancomycin resistance in E. faecium (n = 68) was 47%. The reported results are congruent with global estimates of antibiotic resistance and consumption, providing an overview that could generate actions for antimicrobial optimization at the local and regional levels.

A Five-Year Analysis of Antibiotic Resistance Trends among Bacteria Identified in Positive Urine Samples in a Tertiary Care Hospital from Bucharest, Romania.

The rise of multidrug-resistant bacteria (MDR) has resulted in limited treatment options and poorer outcomes for patients. The objective of this study was to analyze the overall antibiotic resistance trends and distribution for pathogens identified in urine samples at the National Institute of Infectious Diseases "Prof. Dr. Matei Balș" from Bucharest, Romania, over a 5-year period. Antibiotic susceptibility testing was performed using automatic systems and the disk diffusion method. ESBL- and carbapenemases-producing strains were identified using immunochromatography tests, and ROSCO Diagnostica kits were used for definitive confirmation. All results were interpreted according to EUCAST clinical breakpoints. Gram-negative rods (GNR) had overall resistance rates higher than 50% for penicillin and 40% for 3rd- and 4th-generation cephalosporins. Escherichia coli resistance to fosfomycin (3%) and nitrofurantoin (2%) remains low, and 33.30% of E. coli, 48% of Klebsiella spp., and 37% of Pseudomonas aeruginosa isolates were multidrug-resistant (MDR). All Acinetobacter baumannii isolates were MDR by the last year of the study. For Gram-positive cocci (GPC), 37% of all Enterococcus faecium strains and 2% of Enterococcus faecalis were vancomycin-resistant (VRE). E. coli's incidence in UTIs' etiology is on a downward trend. The incidence of Klebsiella spp. and GPCs is rising. Antibiotic stewardship strategies should be implemented after carefully considering regional variations in etiology and resistance trends.

Endoscopic retrograde cholangiopancreatography-obtained bile culture in acute cholangitis: retrospective analysis of bile cultures and risk factors in a tertiary care center.

Collection of bile aspirate during endoscopic retrograde cholangiopancreatography (ERCP) is essential to identify pathogens responsible for acute cholangitis. Limited data are available on the risk factors for the presence of multidrug-resistant organisms (MDRO) in bile. We conducted this retrospective, single-center study to assess the prevalence and susceptibility rates of bacteria in bile cultures, and the risk factors for the presence of pathogens, MDRO, and fungi in bile. All consecutive patients who underwent biliary drainage for acute cholangitis from January 2017 to December 2019 were included. 443/1610 ERCPs were performed for acute cholangitis. Bile culture was collected in 91.4% (405/443), of which 86.7% were positive. Most common isolates were Enterococcus faecalis (37.6%) and Escherichia coli (32.8%). Vancomycin resistance was found in 9.9% of Enterococcus species (spp.); extended-spectrum beta-lactamases (ESBL) and carbapenemases in 11.2% and 0.9% of Enterobacteriaceae, respectively. The empiric antimicrobial therapy was changed in 26.4% (n=107) of cases, with a clinical response in 90.7%. In multivariate analysis, biliary stenting was an independent risk factor for positive bile culture (odds ratio [OR] 9.43; P<0.01). Independent risk factors for MDRO in bile were patient age>60years (OR 2.51; P=0.03), previous sphincterotomy (OR 2.57; P=0.02), and biliary stenting (OR 2.80; P<0.01). Previous sphincterotomy was the only risk factor for isolation of fungi in bile (OR 1.61; P=0.04). Our study showed an increasing prevalence of Enterococcus spp. and MDRO. Bile cultures should be routinely collected in cholangitis and in patients with repeated ERCPs to allow more efficient antimicrobial treatment.

AgrA directly binds to the promoter of vraSR and downregulates its expression in Staphylococcus aureus.

Staphylococcus aureus is an important human pathogen and vancomycin is widely used for the treatment of S. aureus infections. The global regulator agr is known as a well-described virulence regulator. Previous studies have found that agr-dysfunction strains are more likely to develop into vancomycin-resistant strains, but the mechanism for this phenomenon remains unknown. VraSR is a two-component regulatory system related to vancomycin resistance. In this study, we found that the expression levels of vraR were higher in agr-dysfunction clinical strains than in the agr-functional strains. We knocked out agr in a clinical strain, and quantitative reverse transcription PCR and β-galactosidase activity assays revealed that agr repressed transcription of vraR. After vancomycin exposures, population analysis revealed larger subpopulations displaying reduced susceptibility in agr knockout strain compared with wild-type strain, and this pattern was also observed in agr-dysfunction clinical strains compared with the agr-functional strains. Electrophoretic mobility experiment demonstrated binding of purified AgrA to the promoter region of vraR. In conclusion, our results indicated that the loss of agr function in S. aureus may contribute to the evolution of reduced vancomycin susceptibility through the downregulation of vraSR.

Role of membrane vesicles in the transmission of vancomycin resistance in Enterococcus faecium

Clonal transmission and horizontal gene transfer (HGT) contribute to the spread of vancomycin-resistant enterococci (VRE) in global healthcare. Our study investigated vesiduction, a HGT mechanism via membrane vesicles (MVs), for vanA and vanB genes that determine vancomycin resistance. We isolated MVs for VRE of different sequence types (STs) and analysed them by nanoparticle tracking analysis. Selected MV samples were subjected to DNA sequence analysis. In resistance transfer experiments, vancomycin-susceptible enterococci were exposed to MVs and bacterial supernatants of VRE. Compared to bacteria grown in lysogeny broth (MVs/LB), cultivation under vancomycin stress (MVs/VAN) resulted in increased particle concentrations of up to 139-fold (ST80). As a key finding, we could show that VRE isolates of ST80 and ST117 produced remarkably more vesicles at subinhibitory antibiotic concentrations (approx. 9.2 × 1011 particles/ml for ST80 and 2.4 × 1011 particles/ml for ST117) than enterococci of other STs (range between 1.8 × 1010 and 5.3 × 1010 particles/ml). In those MV samples, the respective resistance genes vanA and vanB were completely verifiable using sequence analysis. Nevertheless, no vancomycin resistance transfer via MVs to vancomycin-susceptible Enterococcus faecium was phenotypically detectable. However, our results outline the potential of future research on ST-specific MV properties, promising new insights into VRE mechanisms.

Antibiotic resistance profile in sediments and bacterial isolates from a human-impacted mangrove from Bahía Magdalena, Mexico

Abstract Antibiotic-resistant bacteria (ARB) and antibiotic-resistance genes (ARGs) in the environment are considered emerging contaminants. One of the ecosystems that can harbor resistant bacteria and genes is mangroves. This report aims to assess ARB and ARG's presence at two mangrove locations in Bahia Magdalena, Mexico. Bacterial isolates were selected based on their phenotypic profiles of antibiotic resistance. DNA from mangrove sediments and bacterial isolates was tested for int1 and antibiotic resistance genes by final point PCR. From 90 ARB isolates, the phenotypic profile showed resistance to ciprofloxacin (90%), cefotaxime (85.7%), and ampicillin (78.6%), while the beta-lactamase gene (blaCTX-M) was present in 72.9% of the isolates. Antibiotic resistance genes identified in sediments were found even at 30–50 cm depth, but the integron Class gen (int1) was found mainly in shallow samples. Principal component analysis showed a close relationship between the integron gene, vancomycin, and sulfonamides resistance genes. No correlation was found between the phenotypic and genotypic profiles of the bacterial isolates. Also, no differences were observed between the two mangrove ecosystems studied. The impact of anthropogenic activities was equally significant on both sites. The role of mangrove ecosystems in the dispersion of antibiotic resistance needs to be further explored.

Integration of vanHAX downstream of a ribosomal RNA operon restores vancomycin resistance in a susceptible Enterococcus faecium strain

During the genomic characterisation of Enterococcus faecium strains (n = 39) collected in a haematology ward, we identified an isolate (OI25), which contained vanA-type vancomycin resistance genes but was phenotypically susceptible to vancomycin. OI25 could revert to resistance when cultured in the presence of vancomycin and was thus considered to be vancomycin-variable. Long-read sequencing was used to identify structural variations within the vancomycin resistance region of OI25 and to uncover its resistance reversion mechanism. We found that OI25 has a reduced ability to positively regulate expression of the vanHAX genes in the presence of vancomycin, which was associated with the insertion of an IS6-family element within the promoter region and the first 50 bp of the vanR gene. The vancomycin-resistant revertant isolates constitutively expressed vanHAX genes at levels up to 36,000-fold greater than OI25 via co-transcription with a ribosomal RNA operon. The vancomycin-resistant revertants did not exhibit a significant growth defect. During VRE outbreaks, attention should be paid to contemporaneous vancomycin-susceptible strains as these may carry silent vancomycin resistance genes that can be activated through genomic rearrangements.