The culmination of multidrug-resistant efflux pumps vs. meager antibiotic arsenal era: Urgent need for an improved new generation of EPIs.

Efflux pumps function as an advanced defense system against antimicrobials by reducing the concentration of drugs inside the bacteria and extruding the substances outside. Various extraneous substances, including antimicrobials, toxic heavy metals, dyes, and detergents, have been removed by this protective barrier composed of diverse transporter proteins found in between the cell membrane and the periplasm within the bacterial cell. In this review, multiple efflux pump families have been analytically and widely outlined, and their potential applications have been discussed in detail. Additionally, this review also discusses a variety of biological functions of efflux pumps, including their role in the formation of biofilms, quorum sensing, their survivability, and the virulence in bacteria, and the genes/proteins associated with efflux pumps have also been explored for their potential relevance to antimicrobial resistance and antibiotic residue detection. A final discussion centers around efflux pump inhibitors, particularly those derived from plants.

Distribution of virulence genes and SCCmec types among methicillin-resistant Staphylococcus aureus of clinical and environmental origin: a study from community of Assam, India.

OBJECTIVE: This study was designed to discover the dissemination of virulence genes in Methicillin-resistant Staphylococcus aureus from clinical, community and environmental settings. RESULTS: This study includes 1165 isolates collected from hospital, community and environmental settings. Among them sixty three were confirmed as MRSA with varied SCCmec types viz; type I, type II, type III, type IV, type V, type VI, type VII, type VIII and type XII. The virulence gene such as sea (n = 54), seb (n = 21), eta (n = 27), etb (n = 2), cna (n = 24), ica (n = 2) and tst (n = 30) was also revealed from this study. The study underscores coexistence of resistance cassette and virulence genes among clinical and environment isolates which is first of its kind from this part of the world.

Expressional Pattern of psm-mec System in Methicillin-Resistant Staphylococcus aureus Under Oxacillin Stress.

The psm-mec element and other regulatory factors such as sarA, agrA, and RNAIII are responsible for maintaining the genetic framework for enhanced virulence of MRSA. psm-mec is found predominantly in the staphylococcal cassette chromosome (SCCmec). sarA, agrA, and RNAIII control gene expression to facilitate adaptation in certain environment. Genome-wide approaches have shown that expression of virulence factors is frequently regulated at transcriptional, translational level, and mRNA degradation level. In this study, transcriptional responses of psm-mec gene in accordance with other regulatory factors sarA, agrA, and RNAIII were observed under normal conditions as well as when exposed to 2 µg/ml and 6 µg/ml of oxacillin stress. One-way t-test was carried out for analysing RQ values obtained through real-time PCR. This study showed downregulation of psm-mec gene and upregulation of other regulatory genes at lower concentration of oxacillin. However, this was reverse when exposed against higher concentration of oxacillin. It was observed from the study that the expression of virulence factors were dependent on each other under different concentration of oxacillin. Thus, this study highlights that psm-mec, sarA, agrA, and RNAIII gene are under direct control of antibiotic pressure in a concentration-dependent manner.

Use of fluorescence foldscope as an effective tool for detection of biofilm formation in Pseudomonas aeruginosa.

Purpose: Pseudomonas aeruginosa is an opportunistic pathogen with biofilm-forming ability, by the virtue of which they can evade the immune response and antimicrobial chemotherapy. Several methods have been designed for the detection of biofilms but require sophisticated instrumentation and expertise. The present study, therefore, used an improvised device, 'fluorescence foldscope' which is an origami-based fluorescence microscope as an easy and effective tool to detect biofilm formation. Methodology: Three representatives of P. aeruginosa of clinical origin were taken for the study along with two reference strains PA01 and ATCC27853. The strains were cultured in Luria Bertani (LB) broth with and without carbapenem (imipenem and meropenem) and cephalosporin (ceftazidime, cefotaxime and ceftriaxone) pressure, respectively. The cultures were diluted to 1:100 in LB; seeded with sterile glass slides at 90° angle and incubated for 5 consecutive days. The slides were observed with fluorescence foldscope. Results: Fluorescence emission was observed in two test isolates CD1 and CD2 at 48 and 72 h, respectively, whereas no fluorescence was observed in CD3. The fluorescence observed in the isolates was not affected by 2 µg/ml carbapenem pressure, while with 2 µg/ml ceftazidime stress, a change in fluorescence was observed in CD2 in comparison to the fluorescence observed under normal growth condition. Conclusion: Fluorescence foldscopy is an effective and reliable tool for the detection of biofilm formation in clinical isolates of P. aeruginosa under different laboratory conditions. Biofilm-forming P. aeruginosa worsens the medical condition and is difficult to eradicate. The present study came up with an effective and reliable tool for the detection of biofilm formation in clinical isolates of P. aeruginosa.

Transcriptional response of mar, sox and rob regulon against concentration gradient carbapenem stress within Escherichia coli isolated from hospital acquired infection.

OBJECTIVE: The present study was carried out to investigate the transcriptional response of marA (Multiple antibiotic resistance A gene), soxS (Superoxide S gene) and rob (Right-origin-binding gene) under carbapenem stress. RESULTS: 12 isolates were found over-expressing AcrAB-TolC efflux pump system and showed reduced expression of OmpF (Outer membrane porin) gene were selected for further study. Among them, over expression of marA and rob was observed in 7 isolates. Increasing pattern of expression of marA and rob against meropenem was observed. The clones of marA and rob showed reduced susceptibility towards carbapenems.

Cyathea gigantea (Cyatheaceae) as an antimicrobial agent against multidrug resistant organisms.

BACKGROUND: Rapid emergence of multidrug resistant (MDR) organisms in hospital and community settings often result into treatment failure, thus leading the clinicians with fewer treatment options. Cyathea gigantea, an ethnomedicinally important fern used in cuts and wound infections. So, if this medicinal plant is used in treating the MDR infections then it might bring certain relief in future treatment options. METHODS: Antibacterial activity of C. gigantea against MDR bacteria was assed using well diffusion and broth microdilution methods to determine the diameters of growth inhibition zones, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Synergistic activity was also determined with the conventional antibiotics by disc diffusion method followed by FIC index of each of the tested antibiotic was calculated. The active extract was then subjected to fractionation by column chromatography and antibacterial activity was done with each of the collected fractions. RESULTS: Crude extract of C. gigantea was found to be active against all the tested organisms. The MIC was 200 µg/ml against Gram-positive i.e., Staphylococcus aureus ATCC 25923 and 400 µg/ml against Gram-negative i.e., Escherichia coli ATCC 25922 and Pseudomonas aeruginosa PAO1, while the MBC was 400 µg/ml in case of Gram-positive and 800 µg/ml for Gram-negative. The synergistic activity revealed that the plant extract increased the antibacterial property of the studied antibiotics and the FIC index showed that significant synergistic activity was shown by ciprofloxacin followed by tetracycline, ampicillin and oxacillin. Antibacterial activity with the fractionated extract showed that the FR II, FR III and FR IV were active against both Gram-positive and Gram-negative bacteria, whereas FR I, FR V and FR VI did not show antibacterial property against any of the tested bacteria. CONCLUSIONS: Extracts of C. gigantea was found active against both selected Gram-positive and Gram-negative organisms and thus offers the scientific basis for the traditional use of the fern. The present study also provides the basis for future study to validate the possible use against multidrug resistant organisms.

An array of multiplex PCR assays for detection of staphylococcal chromosomal cassette mec (SCCmec) types among staphylococcal isolates.

Staphylococcal Cassette Chromosome mec (SCCmec) element plays a key role in development of resistance by carrying different resistance factors. Therefore, routine and rapid diagnostic approach is considered advantageous for the easy detection of SCCmec elements. So, here we have described the use of three sets of multiplex PCR assay, which can be used to identify SCCmec type I to type XII, unlike other known protocols. MRSA isolates of both hospital and community settings had been utilized to confirm the sensitivity of the method. All the isolates were examined for SCCmec types using multiplex PCR assay followed by sequencing of amplified products. The results confirmed the detection of SCCmec type I, type II, type III, type IV, type V, type VI, type VII, type VIII and type XII, where SCCmec type II having ST1551 and type V with ST2416 were found to be associated with multidrug resistance and were highly prevalent in the study area. This method will be useful for epidemiological assessment as it will be easier to track the resistance among staphylococci for control of infections and its management.

AcrAB-TolC efflux pump system plays a role in carbapenem non-susceptibility in Escherichia coli.

BACKGROUND: Efflux pump mediated antibiotic resistance is an unnoticed and undetected mechanism in clinical microbiology laboratory. RND efflux systems are known for aminoglycoside and tetracycline resistance whereas their role in carbapenem non-susceptibility is not established. The study was undertaken to investigate the role of efflux pump in providing resistance against carbapenems and their response against concentration gradient carbapenem stress on the transcriptional level of the AcrAB gene in the clinical isolates of Escherichia coli from a tertiary referral hospital of Northeast India. RESULTS: Out of 298 non-susceptible Escherichia coli isolates 98 isolates were found to have efflux pump mediated carbapenem non-susceptibility. Among them thirty-five were non carbapenemase producers and their expressional levels were verified using qRT-PCR under concentration gradient carbapenem stress. In this study, a strong correlation between ertapenem resistance and AcrA overexpression was observed which has not been reported previously. Further, it was observed that imipenem stress increased AcrB expression in Escherichia coli which holds the novelty of this study. Additionally, the transcription of AcrR was insistently increased which is much higher than the transcriptional level of AcrA under concentration gradient carbapenem stress condition. CONCLUSION: The study established that AcrAB pump is a relevant antibiotic resistance determinant in bacterial pathogen, has an important role in developing resistance against carbapenem group of antibiotics.

Effect of concentration gradient carbapenem exposure on expression of blaNDM-1 and acrA in carbapenem resistant Escherichia coli.

Escherichia coli, one of the major pathogens, frequently exhibits carbapenem resistance. It would be of interest to investigate which of the mechanisms responds when a strain that carries both blaNDM-1 and over expressed AcrAB-TolC efflux pump system is exposed against carbapenem under differential concentration gradient stress. Four different sets of strains were used in the study; (i) Strain that have blaNDM-1 and over expressed AcrAB-TolC system (ii) Strain that harbour blaNDM-1 and express AcrAB-TolC at basal level (iii) the strain that is devoid of blaNDM-1 but having over expressed AcrAB-TolC systems and (iv) E. coli AG100A (ΔAcrAB) and E. coli HUE 1 (ΔAcrAB-TolC) where blaNDM-1was cloned. The Quantitative Real time PCR showed blaNDM-1 was over expressed under meropenem and imipenem stress irrespective of concentration gradient. In case of ertapenem, at lower concentration AcrA were over expressed whereas, at higher concentration blaNDM-1 showed elevated expression. A consistent elevated expression of AcrA and AcrB was observed against all carbapenems in the strains devoid of blaNDM-1 where as in case of the strain with basal level expression of AcrA, no significant over expression could be observed for blaNDM-1. In case of clones in group IV, expression of blaNDM-1 was elevated in the presence of carbapenem stress.

Transcriptional response of OmpC and OmpF in Escherichia coli against differential gradient of carbapenem stress.

OBJECTIVE: This study was designed to investigate the transcriptional response of OmpF and OmpC along with an antisense RNA, MicF under concentration gradient carbapenem exposure. RESULT: An elevation in the expression of OmpF gene under concentration gradient imipenem stress from a particular concentration was observed. For OmpC gene a significant decrease in the expression was noticed under concentration gradient imipenem and meropenem stress. The study showed reduction in the expression of OmpC gene against imipenem and meropenem possibly preventing the entry of carbapenem antibiotic inside the cell indicating a possible role in carbapenem resistance.

Detection and molecular typing of methicillin-resistant Staphylococcus aureus from northeastern part of India.

BACKGROUND: In Staphylococcus aureus, methicillin resistance is exhibited by modifications in penicillin-binding protein that minimises the binding affinity to beta-lactam antibiotics. The present study investigated the occurrence of methicillin-resistant S. aureus (MRSA) in community-acquired infections, that is, community-acquired MRSA (CA-MRSA) and in-hospital-acquired infections, that is, hospital-acquired MRSA (HA-MRSA) from Northeast India. METHODS: A total of 197 consecutive non-duplicate isolates were collected from Silchar Medical College and Hospital and other private diagnostic laboratories. The isolates were confirmed to be S. aureus at our centre. All isolates were subjected to antibiotic susceptibility testing and were screened for methicillin resistance using cefoxitin disc test. All MRSA were subjected to Polymerase Chain Reaction (PCR) assay for detection of mecA and mecC genes. DNA fingerprinting was performed for determining clonal diversity. RESULTS: Seventy-one isolates of 127 confirmed S. aureus were found to be methicillin resistant by screening test. mecA gene was detected in 43 isolates, and none of the isolates were positive for mecC gene. Linezolid and teicoplanin showed better activity with susceptibility pattern being 83.6% and 72.44%, respectively, whereas 66.14% were sensitive to vancomycin. Other antibiotic showed low level of activity. Pulsed Field Gel Electrophoresis (PFGE) showed 14 different banding patterns that suggest isolates were of different clonal types. CONCLUSION: mecA was responsible for methicillin resistance in majority of strains. Polyclonal spread of MRSA infection in the study area indicates its diverse origin and possible lateral transfer. Thus, this study is of clinical interest in terms of selection of proper antimicrobial chemotherapy and infection control management.

Association of glycerol kinase gene with class 3 integrons: A novel cassette array within Escherichia coli.

BACKGROUND: Integrons are genetic elements which are known for their role in capturing and spreading of antibiotic resistance determinants among Gram-negative bacilli. So far, there is no study regarding Class 3 integron and their genetic organisation in India. OBJECTIVE: This study investigates the occurrence of Class 3 integron and their gene cassette array among Escherichia coli. MATERIALS AND METHODS: In this study, a total of 200 E. coli isolates were collected from indoor and outdoor patients from Silchar Medical College and Hospital during September 2015 to February 2016. Detection of the integrase genes and gene cassettes within the Class 3 integron was performed by polymerase chain reaction which was further analysed by sequencing. RESULTS: Twenty-seven isolates were found to harbour Class 3 integron. Sequencing of the gene cassettes and whole Class 3 integron revealed the presence of nine different types of cassettes array, out of which the arrangement with glycerol kinase gene cassette was found to be the most prevalent. Arrangement with blaCTX-Mgene cassette was also detected in few isolates. CONCLUSION: This study provides epidemiological profiling of Class 3 integrons in this geographical area. The data generated in this study are helpful in infection control programme, anti-infective research and search for epidemiological markers.

Observation of a new pattern of mutations in gyrA and parC within Escherichia coli exhibiting fluroquinolone resistance.

Therapeutic options with quinolones are severely compromised in infections caused by members of Enterobacteriaceae family. Mutations in chromosomal region are one of the major reasons for bacterial resistance towards this group of antibiotic. The aim of the study is to detect the mutations in gyr A and par C responsible for quinolone resistance among clinical isolates of Escherichia coli. A total of 96 quinolone-resistant clinical isolates of E. coli were collected from a tertiary care hospital of North-east India during March 2015 to August 2015. All the quinolone-resistant E. coli strains were investigated for mutations in the topoisomerases genes gyrA and parC by amplifying and sequencing the quinolone resistance determining regions. Among the 96 E. coli isolates, 83.3% were resistant to nalidixic acid and 80.2%, 66.6%, 23.9% and 50% to ciprofloxacin, norfloxacin, levofloxacin and ofloxacin, respectively. Several alterations were detected in gyrA and parC genes. Three new patterns of amino acid substitution are reported in E. coli isolates. The findings of this study warrant a review in quinolone-based therapy in this region of the world to stop or slow down the irrational use this drug.

Occurrence of II and V staphylococcal cassette chromosome mec types among coagulase-negative staphylococci from Northeastern part of India.

INTRODUCTION: Coagulase Negative Staphylococci, the most commonly isolated pathogen are becoming emerging threats to the community as well as to the nosocomial environment. The present study underscores the distribution of Staphylococcal cassette chromosome mec (SCCmec) types among Methicillin resistant Coagulase Negative Staphylococci from the environmental origin. METHODS AND MATERIALS: Environmental and food sample (n = 460) from different location of northeastern region of India were collected for a period of one year and were phenotypically and genotypically screened using cefoxitin disc and PCR techniques for mecA and mecC gene detection. All the MR-CoNS isolates possessing mecA gene were subjected to 16srDNA sequencing for species identification. SCCmec typing was determined by evaluating using primer sets from type I to type V. Antibiotic susceptibility testing was performed for all the isolates. Statistical analysis with chi-square test using SPSS-21 statistical software. RESULTS: Methicillin resistance shown by one hundred forty three isolates were carried out for molecular analysis, among them 53.84% serves as mecA carrier. Distribution of Staphylococcus haemolyticus was more frequent and was found that SCCmec types II and V were predominant among the study isolates. Linezolid was the drug of choice for the CoNS isolates. Statistical analysis showed an insignificant result for the tested antibiotics and SCCmec types. CONCLUSION: This study therefore interprets the relative importance of SCCmec types among MR-CoNS isolates.

Sub-inhibitory concentration of ertapenem induces overexpression of regulator of antibiotic resistance A in Escherichia coli.

AcrAB-TolC is a tripartite efflux pump system constitutively expressed which functions as an intrinsic-resistant mechanism found to be responsible for conferring resistance towards dyes, detergents and different compounds including various classes of antibiotics. One global regulator belonging to AraC-type regulator family, regulator of antibiotic resistance A (RarA) up-regulates the expression of AcrAB-TolC encoded in Klebsiella pneumoniae, Enterobacter sp. 638, Serratia proteamaculans 568 and Enterobacter cloacae resulting in multidrug-resistant phenotypes. The present work was initiated to find out the transcriptional response of RarA in clinical isolates of Escherichia coli against concentration gradient carbapenem stress. A total of 22 clinical isolates of E. coli and expression level of regulators were analysed via quantitative real-time polymerase chain reaction with and without carbapenem stress. As a result, a strong correlation between the expressional levels of RarA in AcrAB overexpressed isolates of E. coli and elevated expression was observed when exposed under concentration gradient ertapenem stress. The clones containing pRar showed reduction in the zone of inhibition towards carbapenem, indicating the active participation of RarA in AcrAB overexpressed isolates of E. coli conferring resistance towards carbapenems.

Transcriptional response of AcrEF-TolC against fluoroquinolone and carbapenem in Escherichia coli of clinical origin.

INTRODUCTION: Efflux pump systems constitute a major means of intrinsic resistance in Escherichia coli. AcrEF-TolC pump is known to exhibit higher expression level in quinolone resistant isolates. However, the transcriptional response of this pump is yet to be known when exposed to quinolone and other group of antibiotics. OBJECTIVE: The present study analyses the transcriptional response of AcrEF-TolC in the presence of quinolones and carbapenems. METHODOLOGY: A total of 167 non-duplicate clinical isolates from Silchar medical college and Hospital, Silchar, India were included in this study. Of which 27 were devoid of any carbapenemase activity and among them 13 isolates showed overexpression of AcrE and AcrF gene. Transcriptional response of AcrE was directly proportional to increasing concentration of levofloxacin and ofloxacin. However, the response of AcrE and AcrF was inconsistent with carbapenems. RESULT: The study isolates showed susceptibility towards amikacin (68.4%), gentamicin (59.6%), cefepime (52.7%) and pipercillin/tazobactam (48.3%). The present investigation highlights that apart from qnr genes and mutational changes in gyr region, AcrEF-TolC plays a major role in fluoroquinolone resistance in this part of the world. CONCLUSION: Upregulation of AcrE in the presence of levofloxacin and ofloxacin warrants further investigation to establish their active role in efflux of this drug.

Occurrence of Acquired 16S rRNA Methyltransferase-Mediated Aminoglycoside Resistance in Clinical Isolates of Enterobacteriaceae within a Tertiary Referral Hospital of Northeast India.

The methylation of a ribosomal target leads to a high level of resistance to all clinically relevant aminoglycoside antibiotics, so early detection of these resistance determinants will help to reduce the incidence of treatment failures as well as lessen the dissemination rate. Here, we characterized different 16S rRNA methyltransferases responsible for aminoglycoside resistance and their epidemiological background in clinical isolates of Enterobacteriaceae in a tertiary referral hospital in India. All aminoglycoside-resistant isolates were screened for different 16S rRNA methyltransferases by PCR assay, and incompatibility typing of the conjugable plasmid harboring resistance genes was performed by PCR-based replicon typing. An assay for the stability and elimination of these resistance plasmids was performed. The coexistence of extended-spectrum ß-lactamases and metallo-ß-lactamases was also detected, and the heterogeneity of these isolates was determined by enterobacterial repetitive intergenic consensus PCR. The PCR assay revealed the presence of armA, rmtA, rmtB, rmtC, and rmtD in single and multiple combinations, and these were carried by a diverse group of Inc plasmids. Plasmids harboring these resistance determinants were highly stable and maintained until the 55th serial passage, but SDS treatment could easily eliminate the plasmids harboring the resistance determinants. The coexistence of blaTEM, blaPER, blaGES, and blaSHV, as well as blaVIM and blaNDM, within these isolates was also detected. Strains with different clonal patterns of aminoglycoside resistance were found to spread in this hospital setting. We observed that the 16S rRNA methyltransferase genes were encoded within different Inc plasmid types, suggesting diverse origins and sources of acquisition. Therefore, the present study is of epidemiological importance and can have a role in infection control policy in hospital settings.