Molecular Confirmation of Vancomycin-Resistant Staphylococcus aureus with vanA Gene from a Hospital in Kathmandu

Staphylococcus aureus, a commensal on the skin and in the nasal cavity of humans, is one of the most serious cases of nosocomial infections. Moreover, methicillin-resistant S. aureus (MRSA) is a leading cause of morbidity and mortality worldwide. For the treatment of MRSA infections, vancomycin is considered as a drug of choice. However, the emergence of vancomycin resistance among MRSA isolates has been perceived as a formidable threat in therapeutic management. To estimate the rate of vancomycin-resistant S. aureus (VRSA) and to detect the vancomycin-resistant genes, namely, vanA and vanB, among the isolates, a hospital-based cross-sectional study was conducted from July to December 2018 in Annapurna Neurological Institute and Allied Science, Kathmandu, Nepal. S. aureus was isolated and identified from different clinical samples and processed for antibiotic susceptibility testing by the modified Kirby–Bauer disc diffusion method. The screening of MRSA was performed as per Clinical and Laboratory Standard Institute (CLSI) guidelines. VRSA was confirmed by the minimum inhibitory concentration (MIC) method by employing E-test strips. All the phenotypically confirmed VRSA were further processed to detect the vanA and vanB gene by using the conventional polymerase chain reaction (PCR) method. A total of 74 (20.3%) S. aureus were isolated, and the highest percentage of S. aureus was from the wound samples (36.5%). Of 74 S. aureus isolates, the highest number (89.2%) was resistant to penicillin, and on the other hand, linezolid was found to be an effective drug. Likewise, 45 (60.81%) were found to be MRSA, five (11.11%) were VRSA, and 93.2% of S. aureus isolates showed an MAR index greater than 0.2. Two VRSA isolates (40%) were positive for the vanA gene. The higher prevalence of MRSA and significant rate of VRSA in this study recommend routine surveillance for the MRSA and VRSA in hospital settings before empirical therapy.

Identification of Enterococcus faecalis in Different Clonal Lineages with the Pulsed-Field Gel Electrophoresis Method in Imam Khomeini Hospital, Ilam, Iran

Background: Due to the importance of identifying the source of infectious agents, different typing methods have been developed, among which the pulsed-field gel electrophoresis (PFGE) method is known as the gold standard for bacteria. Also, Enterococcus faecalis is classified as a nosocomial infection. Objectives: The current study aimed to identify the source of E. faecalis by the PFGE method. Methods: Bacteria were collected from all cases of urinary tract infections. Then, the identification process was performed. All isolates were evaluated for vancomycin resistance, and then PFGE was carried out. Results: The results of disk diffusion showed that 54% of the isolates showed resistance to vancomycin. Also, 4% of the isolates were intermediate, and 42% showed sensitivity to vancomycin. Afterwards, the PCR of the VanA gene was performed to confirm the results of disk diffusion. Thus, 48 out of 54 (88.8%) isolates had the VanA gene, and none of the four intermediate isolates had the VanA gene. Our results demonstrated that 54 isolates were vancomycin-resistant, and 50 different pulsotypes groups were identified. Conclusions: Our findings showed the isolates of E. faecalis were from different clonal lineages.

Examining Waterways Near UF Campus for Antimicrobial Resistance

The use of clinical antibiotics has greatly increased the scope and severity of naturally occurring antimicrobial resistance (AMR) patterns. Processing of wastewater by wastewater treatment plants (WWTPs) reduces but does not eliminate the concentration of clinical antibiotics present. Bacterial communities residing in aquatic biofilms are more easily able to exchange genes than their planktonic counterparts and thus display a higher rate of AMR. Previous work has indicated the presence of clinical antibiotics and AMR genes in several aquatic systems worldwide. Gainesville, FL has a significant population density and is home to the state’s third-largest hospital as well as a major university; thus, environmental monitoring for AMR genes is a pertinent undertaking. Biofilms were sampled from sites along the Sweetwater Branch Creek upstream and downstream of the GRU Main Street Water Reclamation Facility (WWTP). Colonies were screened for resistance on R2A plates containing ampicillin, methicillin, or vancomycin. After four days, colonies were randomly selected for genetic analysis via polymerase chain reaction (PCR) and gel electrophoresis to detect corresponding antimicrobial resistance genes. Only two colonies of thirty-five were positive for AMR genes: one colony from the downstream site which displayed the ampC gene for ampicillin resistance and one upstream isolate which displayed the vanA gene for vancomycin resistance. The preliminary results of this sampling project indicate only a small amount of resistance present along the Sweetwater Branch Creek, though AMR dynamics may be better illuminated by further analysis.

Identification and characterization of vancomycin-resistant Staphylococcus aureus in hospital wastewaters: evidence of horizontal spread of antimicrobial resistance

Antibiotic resistance has become a major threat to human health around the world, but its spread through the aquatic environment has been often overlooked. This study aimed to determine the occurrence of vancomycin-resistant Staphylococcus aureus in hospital wastewaters and their transmission into public water bodies in Kerala, India. A total of 113 S. aureus were isolated from three hospital effluents in Kerala, India. Standard disc diffusion and the strip method were used for antibiotic susceptibility testing and minimum inhibitory concentration detection. Plasmid-mediated vancomycin resistance was confirmed by plasmid curing and conjugation; resistant genes were detected by the polymerase chain reaction (PCR). Nearly 76% of S. aureus isolates were resistant to β-lactams, chloramphenicol, macrolides, aminoglycosides, and glycopeptide class of antibiotics. Among the vancomycin-resistant Staphylococcus aureus (VRSA) isolates, the prevalence rates of vanA and vanB resistance-encoding genes were 46.5 and 59.3%, respectively. Through the broth mating method, vanA gene was successfully transferred from VRSA donor to vancomycin-sensitive S. aureus. The study strongly indicates the contamination of water bodies with antibiotic-resistant bacteria from hospital discharges, their dissemination and possible transfer to microbes in the aquatic environment, posing a serious threat for public health.

Identification of Vancomycin Resistance Determinants in Twin Cities of Rawalpindi/Islamabad

Vancomycin resistant Enterococci have emerged rapidly in the recent years leading to treatment failure. The aim of this work was to identify vancomycin resistance determinants; vanA and vanB genes in Enterococci. The blood, urine and throat samples were collected from 150 patients from local hospitals of twin cities of Rawalpindi/Islamabad. Forty-nine phenotypically confirmed isolates were further confirmed by PCR amplification for vancomycin determinants (genes for vanA and vanB) for vancomycin resistant Enterococci. Dependence and frequency distribution of VRE and VSE bacteraemia with respect to age, gender and source was also studied. Phenotypically resistant strains were positive for vanA while negative for vanB. vancomycin susceptible enterococci (VSE) could be isolated more from urine samples as compared to blood whereas vancomycin resistant enterococci (VRE) was found more prevalent in the blood samples (p value= 0.000). VRE was more frequently isolated from patients aged 50 or above whereas VSE prevalence was same in both age groups (p value=0.002). Gender was not found to have any significant impact on VRE or VSE bacteraemia. This study reports vanA gene cluster responsible for resistance in Pakistani population and frequently isolation of VRE from blood samples. Keywords: Enterococcus, Vancomycin resistant Enterococci, Pakistan

Effect of vancomycin on cytoplasmic peptidoglycan intermediates and van operon mRNA levels in VanA-type vancomycin resistant Enterococcus faecium

Resistance in VanA-type vancomycin resistant Enterococcus faecium (VREfm) is due to an inducible gene cassette encoding seven proteins ( vanRSHAXYZ ). This provides for an alternative peptidoglycan (PG) biosynthesis pathway whereby d- Alanyl- d- Alanine ( d -Ala- d -Ala) is replaced with d- Alanyl- d- Lactate ( d -Ala- d -Lac), to which vancomycin cannot bind effectively. This study aimed to quantify cytoplasmic levels of normal and alternative pathway PG intermediates in VanA-type VREfm by liquid chromatograph tandem mass spectrometry before and after vancomycin exposure, and to correlate these changes with changes in vanA operon mRNA levels measured by RT-qPCR. Normal pathway intermediates predominate in the absence of vancomycin, with low levels of alternative pathway intermediates. Extended (18 hr) vancomycin exposure resulted in a mix of the terminal normal (UDP-Penta) and alternative (UDP-Pentadepsi) pathway intermediates (2:3 ratio). Time courses reveal normal pathway intermediates responding rapidly (peaking in 3-10 minutes), and alternative pathway intermediates responding more slowly (peaking in 15-45 minutes). RT-qPCR demonstrated that vanA operon mRNA transcript levels increase rapidly after exposure, reaching maximal levels in 15 minutes. To resolve the effect of increased van operon protein expression on PG metabolite levels, linezolid was used to block protein biosynthesis. Surprisingly, linezolid dramatically reduced PG intermediate levels when used alone. When used in combination with vancomycin, linezolid only modestly reduced alternative UDP-linked PG intermediate levels, indicating substantial alternative pathway presence before vancomycin exposure. Comparison of PG intermediate levels between VREfm, vancomycin sensitive Enterococcus faecium (VSEfm), and methicillin resistant Staphylococcus aureus (MRSA) after vancomycin exposure demonstrate substantial differences between S. aureus and E. faecium PG biosynthesis pathways. IMPORTANCE VREfm is highly resistance to vancomycin due to the presence of a vancomycin resistance gene cassette. Exposure to vancomycin induces the expression of genes in this cassette, which encode for enzymes that provide for an alternative peptidoglycan (PG) biosynthesis pathway. In VanA-type resistance these alternative pathway enzymes replace the d -Ala- d -Ala terminus of normal PG intermediates with d -Ala- d -Lac terminated intermediates, to which vancomycin cannot bind. While the general features of this resistance mechanism are well known, the details of the choreography between vancomycin exposure, VanA gene induction, and changes in the normal and alternative pathway intermediate levels has not been described previously. This study comprehensively explores how VREfm responds to vancomycin exposure at the mRNA and PG intermediate levels.

Characterization of Enterococci- and ESBL-Producing Escherichia coli Isolated from Milk of Bovides with Mastitis in Egypt

This study aimed to investigate the prevalence and antimicrobial resistance of enterococci- and ESBL-producing E. coli isolated from milk of bovine mastitis cases in Egypt. Fifty milk samples of dairy animals were collected from localities in the Nile Delta region of Egypt. Isolates were identified using MALDI-TOF MS, and antibiotic susceptibility testing was performed by the broth microdilution method. PCR amplifications were carried out, targeting resistance-associated genes. Seventeen Enterococcus isolates and eight coliform isolates could be cultivated. Vancomycin resistance rate was high in Ent. faecalis. The VITEK 2 system confirmed all E. coli isolates as ESBL-producing. All Ent. faecalis isolates harbored erm(B), tetL and aac-aphD genes. The vanA gene was detected in Ent. faecalis isolate, vanB was found in other Enterococcus, while one isolate of E. casseliflavus exhibited the vanA gene. E. coli isolates exhibited high prevalence of erm(B) and tetL. E. coli isolates were analyzed by DNA microarray analysis. Four isolates were determined by O-serotyping as O8 (n = 1), O86 (n = 2) and O157 (n = 1). H-serotyping resulted in H11, H12, H21 (two isolates each) and one was of H16 type. Different virulence-associated genes were detected in E. coli isolates including lpfA, astA, celB, cmahemL, intI1 and intI2, and the iroN gene was identified by DNA microarray analysis.

Characterization of a vancomycin-resistant Enterococcus faecium isolate and a vancomycin-susceptible E. faecium isolate from the same blood culture

Objectives To characterize two Enterococcus faecium isolates with different resistance phenotypes obtained from the same blood culture. Methods The isolates were identified by MALDI-TOF MS and antimicrobial susceptibility testing (AST) was performed using a VITEK® 2 AST P592 card and Etest. WGS was performed on the MiSeq and MinION sequencer platforms. Core-genome MLST (cgMLST) and seven-loci MLST were performed. Plasmid analysis was performed using S1-PFGE followed by Southern-blot hybridization. Results Both E. faecium isolates were ST203. AST revealed that one was a vancomycin-resistant E. faecium (VREfm) isolate and the other was a vancomycin-susceptible E. faecium (VSEfm) isolate. The VREfm isolate harboured the vanA gene cluster as part of a Tn1546-type transposon encoded on a 49 kb multireplicon (rep1, rep2 and rep7a) plasmid (pAML0157.1). On the same plasmid, ant(6)-Ia, cat-like and erm(B) were encoded. The VSEfm isolate harboured a rep2 plasmid (pAML0158.1), 12 kb in size, which was present in full length as part of pAML0157.1 from the VREfm isolate. The vanA-encoding pAML0157.1 was a chimera of the rep2 pAML0158.1 and a second DNA segment harbouring vanA, ant(6)-Ia, erm(B) and cat-like, as well as the replicons rep1 and rep7a. By cgMLST analysis, the VREfm and VSEfm isolates were identical. Conclusions Our results demonstrate that the VREfm and VSEfm blood culture isolates represented ST203 and were identical. The investigated heterogeneous resistance phenotypes resulted from the acquisition or loss of plasmid segments in the enterococcal isolates. These data illustrate that mobile genetic elements may contribute to the spread of vancomycin resistance among enterococci and to the genotypic and phenotypic variation within clonal isolates.

Nisin Influence on the Antimicrobial Resistance Ability of Canine Oral Enterococci

Periodontal disease (PD) is one of the most common diseases in dogs. Although previous studies have shown the potential of the antimicrobial peptide nisin for PD control, there is no information regarding its influence in the development of antimicrobial resistance or horizontal gene transfer (HGT). Nisin’s mutant prevention concentration (MPC) and selection window (MSW) were determined for a collection of canine oral enterococci. Isolates recovered after the determination of the MPC values were characterized for their antimicrobial profile and its nisin minimum inhibitory and bactericidal concentrations. The potential of vanA HGT between Enterococcus faecium CCGU36804 and nine clinical canine staphylococci and enterococci was evaluated. Nisin MPC values ranged from 400 to more than 600 μg/mL. In comparison with the original enterococci collection, the isolates recovered after the determination of the nisin MPC showed increased resistance towards amoxicillin/clavulanate (5%), vancomycin (5%), enrofloxacin (10%), gentamicin (10%) and imipenem (15%). The HGT of vanA gene was not observed. This work showed that nisin selective pressure may induce changes in the bacteria’s antimicrobial resistance profile but does not influence horizontal transfer of vanA gene. To our knowledge, this is the first report of nisin’s MPC and MSW determination regarding canine enterococci.