Development and Validation of UFLC-MS/MS Analytical Method for the Simultaneous Quantification of Antibiotic Residues in Surface Water, Groundwater, and Pharmaceutical Waste Water Samples from South India.

Antibiotic residues in pharmaceutical wastewater pose a significant environmental concern due to their potential role in fostering antimicrobial resistance. South Indian pharmaceutical companies produce a wide range of antibiotics. As a result, the industries that discharge water may include antibiotic residues, which could be harmful to the environment. In this study, a novel, quick, accurate, and sensitive approach for the simultaneous detection of 11 antibiotics was established, and triple quadrupole mass spectrometry, ultra-fast liquid chromatography-tandem mass spectrometry (UFLC-MS/MS), and selective solid-phase extraction (SPE) were used for validation. Utilizing a mixed mode reversed-phase/cation-exchange cartridge (SPE using Strata X, 33 µm), the single-cartridge extraction procedure was performed and validated. Relative standard deviations for most of the antibiotics ranged from 3.5 to 0.56 with recoveries ranging from 57 to 85%. The samples were injected into the UFLC-MS/MS apparatus at a volume of 10 µL for analysis. The auto sampler cooler temperature was kept at 150 °C, while the column temperature was kept at 40 °C. After validation, the technique was determined to be linear in the range of 2.0-1000.0 ng/mL. The retention period for antibiotics was between 1.2 and 1.5 min. Antibiotics transitions for multiple reaction monitoring| were between 235.1/105.9 and 711.5/467.9 m/z. The method of analysis took 2.5 min to run completely. Antibiotic residues were efficiently analyzed using the established analytical approach in pharmaceutical wastewater (influent and effluent), surface, and groundwater. Eleven antibiotics were found in the water samples during examination with concentrations ranging between 2.313 and 95.744 ng/L. The procedure was shown to be much more environmentally friendly than other contemporary methods based on the green analytical procedure index's evaluation of greenness. Blue applicability grade index tool indicated the developed method's practicality in comparison with that of other reported method.

Spectrum of Virulence Factors in Clinical Isolates of Staphylococcus aureus and Prevalence of SCCmec Types in Methicillin-Resistant Staphylococcus aureus in a Tertiary Care Center.

Background Methicillin-resistant Staphylococcus aureus (MRSA) is a widely recognized multidrug-resistant bacteria presenting a major therapeutic challenge to clinicians. Staphylococcus aureus possesses a number of pathogenicity factors that attribute to the severity of infections. This study was undertaken to investigate the common virulence genes in clinical isolates of Staphylococcus aureus , determine their antimicrobial susceptibility profile, and to characterize the staphylococcal cassette chromosome mec (SCCmec) types among MRSA in a tertiary care center. Materials and Methods A total of 133 clinical isolates were included in this study. Susceptibility to various antibiotics was determined by disc diffusion method. Methicillin resistance was screened using cefoxitin disc; m ecA and mecC genes were detected using polymerase chain reaction (PCR). PCR was done to detect 12 virulence factors such as hla , hlb , fnbA , fnbB , sea , seb , sec , icaA , clfA , tst , pvl, and eta . SCCmec typing was done by multiplex PCR. Results Of the 133 clinical isolates, 54 (40.6%) were MRSA. The most common virulence gene detected was hlb (61.6%), hla (39%), and fnbA (37%). SCCmec type I was the most predominant. Mortality rate of 6.7% was observed among patients with staphylococcal infections. Univariate analysis of mortality associated virulence genes did not reveal any significant association between virulence genes and mortality. Conclusion The distribution of virulence genes is similar in both MRSA and methicillin-sensitive Staphylococcus aureus . MRSA belongs to the SCCmec types I to IV. Possession of multiple virulence factors and multidrug resistance profile makes Staphylococcus aureus a formidable pathogen in clinical settings.

Detection of a Novel G2603T Mutation in cfr Harboring Linezolid-Resistant Staphylococcus haemolyticus : First Report from India.

Background Staphylococcus haemolyticus has emerged as an important multidrug-resistant nosocomial pathogen. Linezolid is useful in the treatment of severe infections caused by methicillin-resistant Staphylococci . Resistance to linezolid in Staphylococci is due to one or more of the following mechanisms: acquisition of the cfr (chloramphenicol florfenicol resistance) gene, mutation in the central loop of domain V of the 23S rRNA, and mutation in the rplC and rplD genes. This study was carried out to detect and characterize resistance to linezolid among the clinical isolates of Staphylococcus haemolyticus . Materials and Methods The study included 84 clinical isolates of Staphylococcus haemolyticus . Susceptibility to various antibiotics was determined by disc diffusion method. Minimum inhibitory concentration (MIC) was determined by agar dilution method for linezolid. Methicillin resistance was screened using oxacillin and cefoxitin disc. Polymerase chain reaction was done to detect mecA, cfr and mutations in the V domain of the 23S rRNA gene. Results Resistance to linezolid was exhibited by 3 of the 84 study isolates with MIC more than 128 µg/mL. The cfr gene was detected in all the three isolates. The G2603T mutation was observed in the domain V of the 23S rRNA among two isolates, whereas one isolate lacked any mutation. Conclusion The emergence and spread of linezolid-resistant Staphylococcus haemolyticus isolates carrying G2603T mutation in the domain V of the 23S rRNA and harboring the cfr gene pose a threat in clinical practice.

Plasmid-Mediated Fluoroquinolone Resistance in Pseudomonas aeruginosa and Acinetobacter baumannii.

Introduction Pseudomonas aeruginosa and Acinetobacter baumannii are important pathogens in health care-associated infections. Fluoroquinolone resistance has emerged in these pathogens. In this study, we aimed to determine the occurrence of plasmid-mediated quinolone resistance (PMQR) determinants ( qnrA , qnrB , qnrS , aac(6')-Ib-cr , oqxAB , and qepA ) by polymerase chain reaction (PCR) and the transmissibility of plasmid-borne resistance determinants in clinical isolates of P. aeruginosa and A. baumannii . Materials and Methods The study included P. aeruginosa (85) and A. baumannii (45) which were nonduplicate, clinically significant, and ciprofloxacin resistant. Antibiotic susceptibility testing was done by disk diffusion method for other antimicrobial agents, namely amikacin, ceftazidime, piperacillin/tazobactam, ofloxacin, levofloxacin, and imipenem. Minimum inhibitory concentration of ciprofloxacin was determined. Efflux pump activity was evaluated using carbonyl-cyanide m-chlorophenylhydrazone (CCCP). The presence of PMQR genes was screened by PCR amplification. Transferability of PMQR genes was determined by conjugation experiment, and plasmid-based replicon typing was performed. Results Resistance to other classes of antimicrobial agents was as follows: ceftazidime (86.9%), piperacillin/tazobactam (73.8%), imipenem (69.2%), and amikacin (63.8%). The minimal inhibitory concentration (MIC)50 and MIC90 for ciprofloxacin were 64 and greater than or equal to 256 µg/mL, respectively. There was a reduction in MIC for 37 (28.4%) isolates with CCCP. In P. aeruginosa , 12 (14.1%) isolates harbored qnrB , 12 (14.1%) qnrS , 9 (10.5%) both qnrB and qnrS , 66 (77.6%) aac(6')-Ib-cr , and 3 (3.5%) oqxAB gene. In A. baumannii , qnrB was detected in 2 (4.4%), 1 (2.2%) harbored both the qnrA and qnrS , 1 isolate harbored qnrB and qnrS , 21 (46.6%) aac(6')-Ib-cr , and 1 (2.2%) isolate harbored oqxAB gene. Notably, qepA gene was not detected in any of the study isolates. Conjugation experiments revealed that 12 (9.2%) were transferable. Of the transconjugants, seven (58.3%) belonged to IncFII type plasmid replicon, followed by four (33.3%) IncA/C and one (8.3%) IncFIC type. Conclusion The plasmid-mediated resistance aac(6')-Ib-cr gene is primarily responsible for mediating fluoroquinolone resistance in clinical isolates of P . aeruginosa and A. baumannii . The predominant plasmid type is IncFII.

Molecular Characterization of High-Level Aminoglycoside Resistance among Enterococcus Species.

Background Enterococci are nosocomial pathogen. They can develop high-level resistance to aminoglycoside by producing aminoglycoside modifying enzymes (AMEs). In enterococci, high level resistance to aminoglycosides is mediated by acquisition of plasmid mediated genes encoding for aminoglycoside modifying enzymes (AMEs). High level gentamicin resistance (MIC ≥ 500µg /mL) is predominantly mediated by aac(6')-Ie-aph(2″)-Ia, encoding the bifunctional aminoglycoside modifying enzyme AAC(6')-APH(2″). This enzyme eliminates the synergistic activity of gentamicin when combined with a cell wall active agent. Other AME genes such as aph(2″)-Ib, aph(2″)-Ic, aph(2″)-Id and ant(4')-1a have also been detected in enterococci. Objective This study was carried out to determine the diverse prevalence of AME and their pattern of occurrence in the clinical isolates of Enterococci . Materials and Methods A total number of 150 clinical isolates were included in this study. Susceptibility to various antibiotics was determined by disc diffusion. Minimum Inhibitory Concentration (MIC) was ascertained by agar dilution method. Polymerase chain reaction was done to screen the following AMEs (aac(6')-Ie-aph(2″)-Ia; aph(2″)-Ib; aph(2″)-Ic; aph(2″)-Id and aph(3')- IIIa genes) . Results 51.3% of the study isolates exhibited high level gentamicin resistance. Polymerase chain reaction revealed that aph(3')-111a is the most prevalent AME, followed by aac(6')-1e-aph(2″)-1a . The combination of both the genes were detected in 44.1% of the study isolates. The rest of the AMEs and their combinations were not encountered in this study. 8.6% of the study isolates did not harbour any AME genes screened for, but was phenotypically resistant to gentamicin. In contrast 31.3% anchored the AME genes but phenotypically appeared susceptible to gentamicin. Conclusion This study indicates the high- level aminoglycoside resistance disseminated among Enterococci in our geographical region. It also emphasizes the detection of AMEs by PCR is mandatory because strains that appear susceptible by disc diffusion and/or MIC method may harbour one or more AMEs genes leading to therapeutic failure.

Candida auris, an emerging pathogen - Challenge in the survival of microprimies.

Clinical profile of extreme preterm neonates and more so, of microprimies with birth weight < 800 g is not studied till now. Our article elaborates the profile of 5 microprimies with C.auris sepsis and review of literature. The mean gestational age and birth weight were 26 weeks ± 5 days and 709 ±â€¯64 g respectively. Mortality was 80%. The organism was susceptible to micafungin, voriconazole but was resistant to fluconazole and amphotericin. Among the 5 babies, one had organ involvement in the form of cardiac vegetation. Early identification and optimal choice of drug are crucial for better survival in C.auris sepsis.

Disposable low-cost cardboard incubator for thermoregulation of stable preterm infant - a randomized controlled non-inferiority trial.

BACKGROUND: Incubators and radiant warmers are essential equipment in neonatal care, but the typical 1,500 to 35,000 USD cost per device makes it unaffordable for many units in low and middle-income countries. We aimed to determine whether stable preterm infants could maintain thermoregulation for 48 h in a low-cost incubator (LCI). METHODS: The LCI was constructed using a servo-heater costing 200 USD and cardboard infant-chamber. We conducted this open-labeled non-inferiority randomized controlled trial in a tertiary level teaching hospital in India from May 2017 to March 2018. Preterm infants on full feeds and receiving incubator or radiant warmer care were enrolled at 32 to 36 weeks post-menstrual age. We enrolled 96 infants in two strata (Strata-1< 33 weeks, Strata-2 ≥ 33 weeks at birth). Infants were randomized to LCI or standard single-wall incubator (SSI) after negative incubator cultures and monitored for 48 h in air-mode along with kangaroo mother care. The incubator temperature was adjusted manually to maintain skin and axillary temperatures between 36.5 °C and 37.5 °C. During post-infant period after 48 h, SSI and LCI worked for 5 days and incubator temperatures were measured. The primary outcome was maintenance of skin and axillary temperatures with a non-inferiority margin of 0.2 °C. Failed thermoregulation was defined as abnormal axillary temperature (< 36.5 °C or >37.5 °C) for > 30 continuous-minutes. Secondary outcomes were incidence of hypothermia and required incubator temperature. Trial registration details: Clinical Trial Registry - India (CTRI/2015/10/006316). FINDINGS: Prior to enrollment 79(82%) infants were in radiant warmer and 17(18%) infants were in incubator care. Median weight at enrollment in Strata-1 and Strata-2 for SSI vs. LCI was 1355(IQR 1250-1468) vs. 1415(IQR 1280-1582) and 1993(IQR 1595-2160) vs. 1995(IQR 1632-2237) grams. Mean skin temperature in Strata-1 and Strata-2 for SSI vs. LCI was 36.8 °C ± 0.2 vs. 36.7 °C ± 0.18 and 36.8 °C ± 0.22 vs. 36.7 °C ± 0.19. Mean axillary temperature in Strata-1 and Strata-2 for SSI vs. LCI was 36.9 °C ± 0.19 vs. 36.8 °C ± 0.16 and 36.8 °C ± 0.2 vs. 36.8 °C ± 0.19. Mixed-effect model done for repeated measures of skin and axillary temperatures showed the estimates were within the non-inferiority limit; -0.07 °C (95% CI -0.11 to -0.04) and -0.06 °C (95% CI -0.095 to -0.02), respectively. Failed thermoregulation did not occur in any infants. Mild hypothermia occurred in 11 of 48(23%) of SSI and 16 of 48(33%) of LCI, OR 1.28 (95%CI 0.85 to 1.91). Incubator temperature in LCI was higher by 0.7 °C (95%CI 0.52 to 0.91). In the post-infant period SSI and LCI had excellent reliability to maintain set-temperature with intra-class correlation coefficient of 0.93 (95%CI 0.92 to 0.94) and 0.96 (95%CI 0.96 to 0.97), respectively. INTERPRETATION: Maintenance of skin and axillary temperature of stable preterm infants in LCI along with kangaroo mother care was non-inferior to SSI, but at a higher incubator temperature by 0.7 °C. No adverse events occurred and LCI had excellent reliability to maintained set-temperature. FUNDING: Food and Drug Administration (Award number P50FD004895).

Fluoroquinolone Resistance in Clinical Isolates of Klebsiella Pneumonia e.

Introduction Fluoroquinolones are widely used broad-spectrum antibiotics. Recently, increased rate of resistance to this antibiotic has been observed in Klebsiella pneumoniae. The aim of the present study was to determine the presence of quinolone resistance determining regions (QRDR) mutation genes and plasmid-mediated quinolone resistance (PMQR) determinants in clinical isolates of ciprofloxacin-resistant K. pneumoniae. Material and Methods The study included 110 nonduplicate ciprofloxacin-resistant K. pneumoniae clinical isolates. Antibiotic susceptibility testing by disk diffusion method and minimum inhibitory concentration (MIC) by agar dilution methods for ciprofloxacin was performed according to the recommendations of Clinical Laboratory Standards Institute. The presence of QRDR genes and PMQR genes was screened by polymerase chain reaction (PCR) amplification. Result All 110 isolates were resistance to ciprofloxacin, levofloxacin, and ofloxacin. As much as 88% of the isolates exhibited high-level of MIC to ciprofloxacin. Among the 110 isolates, 94(85%) harbored gyrA and 85 (77%) gyrB. The parC and parE genes were detected in 88 (80%) and 64 (58%) isolates. qnrB was detected in 13 (12%) isolates and qnrS in 5 (4.5%) isolates. Two (1.8%) isolates carried both qnrB and qnrS genes. The acc (6')-Ib-cr gene was found in 98 (89%) isolates and oqxAB was detected in 7 (6.3%) isolates. One (0.9%) isolate carried qnrB, acc(6')-Ib-cr and oqxAB genes. Conclusion The prevalence of acc (6')-Ib-cr gene is high among PMQR determinants, followed by qnrB, oqxAB and qnrS.

Spectrum of Aminoglycoside Modifying Enzymes in Gram-Negative Bacteria Causing Human Infections.

Introduction Aminoglycosides are formidable broad-spectrum antibiotics used in clinical settings; woefully their usage has been reduced by the emergence and distribution of resistance mainly due to aminoglycoside modifying enzymes (AME). Purpose This study was performed to determine the diverse prevalence of AME and their pattern of occurrence in the clinical isolates of gram-negative bacteria. This study also aimed to detect the presence of AMEs that are prevalent in gram-positive bacteria, among gram negatives. Materials and Methods A total number of 386 clinical isolates were included in this study. Polymerase chain reaction revealed the prevalence rate of AMEs screened [aac(6')-lb, aac(3')-I, aac(3')-II, aac(3')-VI, ant(2')-I, ant(4')-IIb, aac(3')-III, aac(3')-IV, aph(2')-Ib, aph(2')-Ic, aph(2')-Id, aac (6')-Ie- aph(2')-Ia, and aph(3')-IIIa]. Conjugation experiment was performed for the clinical isolates which harbored any one of the AME which was prevalent in gram-positive bacteria [aph(3')-IIIa, aac(6')-Ie-aph(2')-Ia]. Results aac(6')-lb is the most prevalent AME, followed by aac(3')-I, aph(3')-VI, aac(3')-VI, and aac(3')-II. The AMEs such as ant (2')-I, ant(4')-IIb, aac(3')-III, aac(3')-IV, aph(2')-Ib, aph(2')-Ic, and aph(2')-Id were not established in our study isolates. The rate of prevalence of aph(3')-IIIa, aac(6')-Ie-aph(2')-Ia-the AMEs encountered in gram-positive and their co-existence was 19.68% and the conjugation experiment revealed their transfer via plasmids. Conclusion This is the first report from India revealing the presence and prevalence of AMEs which are often encountered among gram-positive bacteria in gram negatives and their presence on conjugative plasmids.

Characterisation of virulence genes associated with pathogenicity in Klebsiella pneumoniae.

Purpose: This study was undertaken to characterise the virulence factors in clinical strains of Klebsiella pneumoniae and analyse their association with various infections caused and also to determine the association between virulence factors and antimicrobial resistance profile. Materials and Methods: A total number of 370 clinically significant, non-duplicate isolates of K. pneumoniae isolated from both hospitalised patients and patients attending clinics were included in this study. Polymerase chain reaction (PCR) was carried out for the detection of various virulence genes such as mucoviscosity-associated gene A (magA), gene associated with allantoin metabolism (allS), Klebsiella ferric iron uptake(Kfu), capsule-associated gene A (K2A), regulator of mucoid phenotype A (rmpA), enterobactin (entB), yersiniabactin (YbtS), aerobactin, Fimbrial adhesin (FimH) and uridine-diphosphate galacturonate 4-epimerase (uge). Antimicrobial susceptibility testing and PCR-based detection of beta-lactamase-encoding genes such as extended-spectrum beta-lactamases, AmpCs and carbapenemases were performed. Univariate analysis was done to find the association between virulence genes and mortality. Results: The siderophore, entB, was present in most (90.5%) of the isolates. Of the 370 isolates, 345 carried multiple virulence genes; 15 harboured single virulence genes and 10 did not harbour any of the studied virulence genes. The most common combination of occurrence was entB and FimH. A mortality rate of 12.75% (38/298) was observed among hospitalised patients. None of the virulence genes had any significant association with mortality. Conclusion: Pathogenic K. pneumoniae can harbour single to multiple virulence genes. Invasive infection with even a single virulence gene-harbouring K. pneumoniae can lead to poor outcomes. Both multidrug-resistant (MDR) and non-MDR K. pneumoniae can harbour a variety of virulence genes. None of the virulence genes have a significant association with mortality.

Pulmonary Infections Caused by Emerging Pathogenic Species of Nocardia.

Pulmonary infections are the most common clinical manifestations of Nocardia species. There is an increase in cases of nocardial infections occurring worldwide attributable to the increase in the immunosuppressed population. The availability of molecular methods has aided the detection of more number of cases as well as unusual species. Still, it remains one of the most underdiagnosed pathogens. Recognition of drug resistance in this organism has now mandated early and precise identification with speciation for effective treatment and management. Nocardial species identity can predict antimicrobial susceptibility and guide clinical management. Here, we report two cases of pulmonary nocardiosis caused by unusual species of Nocardia, namely, N. cyriacigeorgica and N. beijingensis identified by 16S rRNA gene-based sequencing. These cases are being reported for their rarity.

Prevalence and clonal relatedness of NDM and OXA-48-producing Klebsiella pneumoniae in a tertiary care hospital in South India.

BACKGROUND: Carbapenems are used for the treatment of serious infections caused by multidrug-resistant Klebsiella pneumoniae. Resistance to carbapenems in K. pneumoniae is mainly due to metallo-beta-lactamases (NDM, IMP, and VIM) and class D oxacillinase (OXA-48-like). AIM AND OBJECTIVE: This study was undertaken to detect the genes encoding for carbapenemase in K. pneumoniae and to determine the clonal relatedness of selected isolates of K. pneumoniae producing NDM and OXA-48 by pulsed-field gel electrophoresis method (PFGE). MATERIALS AND METHODS: The isolates were collected over a period of 1 year. A total of 370 clinically significant, nonduplicate isolates of K. pneumoniae were included in this study. Phenotypic tests for the detection of carbapenemases were performed for all the isolates. Polymerase chain reaction (PCR) was carried out for the detection of carbapenemase genes such as bla KPC, bla IMP, bla VIM, bla NDM, and bla OXA-48. PFGE was performed, and the PFGE profiles were analyzed and compared using BioNumerics version 7.6. RESULTS: Of the 370 isolates of K. pneumoniae, carbapenemase genes were detected in 13.78% (51/370). bla OXA-48 was the prevalent gene detected followed by bla NDM and bla KPC. Thirty strains of K. pneumoniae selected by PFGE analysis were divided into five clusters (A, B, C, D, and E). Cluster C was the major type detected carrying bla NDM and bla OXA-48 genes. CONCLUSION: bla OXA-48 was the most prevalent gene detected in this study. PCR is useful in detecting carbapenemase genes, especially bla NDM, which may show false susceptibility to carbapenems. There was no direct correlation detected between PFGE profiles and antibiotic susceptibility pattern. PFGE has revealed the genomic diversity among isolates, thereby suggesting heterogeneity in strain circulation within intensive care unit and wards of the hospital. Monitoring and molecular typing is essential to curtail the spread of multidrug-resistant strains and control the outbreaks of infection.

Prevalence of blaKPC and its occurrence with other beta-lactamases in Klebsiella pneumoniae.

BACKGROUND: Klebsiella pneumoniae (K. pneumoniae) is an important nosocomial pathogen, and the emergence of multidrug resistance in these organisms limits the treatment options for serious infections caused by them. K. pneumoniae carbapenemase (KPC) is one of the clinically significant Class A beta-lactamases. AIM AND OBJECTIVE: This study was aimed to detect the KPC and its coexistence with other beta-lactamases in K. pneumoniae. MATERIALS AND METHODS: A total of 370 isolates, collected over a period of 1 year, were included in this study. The source of these isolates were urine (n = 170), exudative specimens (n = 132), respiratory secretions such as bronchial wash, endotracheal aspirate, and pleural fluid (n = 38), and blood (n = 30). For all the isolates, antibiotic susceptibility tests by disc diffusion, modified Hodge test, and KPC screening test were done. Polymerase chain reaction (PCR) was performed for the detection of KPC and the copresence of other beta-lactamases genes. RESULTS: Among the 370 isolates, 41 were resistant to the carbapenem by disc diffusion and minimum inhibitory concentration tests. Screen test using ertapenem and the boronic acid disk was positive in 14 isolates. Only one isolate harbored KPC gene by PCR, and it was co-produced with SHV-12 and CTX-M-15. CONCLUSION: PCR remains the gold standard for detection of KPC compared with any other phenotypic methods. Early detection of these genes helps in initiating proper antibiotic treatment.

Occurrence and characterization of hyperviscous K1 and K2 serotype in Klebsiella pneumoniae.

BACKGROUND: Klebsiella pneumoniae causes both nosocomial and community-associated infections. Hypervirulent K. pneumoniae (hvKP), new variant of K. pneumoniae, can cause invasive infections in young healthy individuals as well as in the immunocompromised population. Hypervirulent strains frequently belong to capsular serotypes K1 or K2. Emergence of antimicrobial resistance in hvKP is a cause for concern. AIM AND OBJECTIVE: The present study was done to detect the K1 and K2 serotypes among clinical isolates of K. pneumoniae, spectrum of infections caused by them and presence of common beta-lactamases encoding genes in them. MATERIALS AND METHODS: A total of 370 isolates of K. pneumoniae, isolated from various clinical samples over a period of 1 year was included in this study. Antibiotic susceptibility testing to various classes of antimicrobials was done as per Clinical and Laboratory Standard Institute guidelines. The presence of K2A (specific to serotype K2), magA (specific to serotype K1), and rmpA genes was detected by multiplex polymerase chain reaction (PCR). Extended-spectrum beta-lactamases (TEM, SHV, and CTX-M), plasmid-mediated AmpCs (MOX, CIT, DHA, ACC, EBC, and FOX), and carbapenemase genes (IMP, VIM, NDM, KPC, and OXA-48) were also determined by PCR. RESULTS: Among the 370 isolates, 8 harbored K2A gene and one harbored magA. rmpA gene was detected in three isolates along with K1 or K2 serotypes. Seven K2A-positive isolates were resistant to one or more classes of antimicrobials. The studied ESBL genes were present in four isolates. Two isolates harbored carbapenemase genes (NDM-1, OXA-48) along with ESBLs. CONCLUSION: K2 serotype is more prevalent among hvKP isolates. They can harbor ESBLs and Carbapenemase genes. K1 serotype is rather uncommon in K. pneumoniae. Acquisition of multidrug-resistant genes by these strains adds to their virulence and limits the treatment options.

Enhancement of antimicrobial activity by liposomal oleic acid-loaded antibiotics for the treatment of multidrug-resistant Pseudomonas aeruginosa.

In this study, we examined the efficacy of liposomal oleic acid-based antibiotic formulations on 32 strains of multidrug-resistant Pseudomonas aeruginosa (MDRPa). The average size of liposomes were 93.12 ± 2.3 nm holding a negative zeta potential at -57.3 ± 0.89. Liposomal antibiotic formulations were tested against 32 MDRPa strains isolated from burn wounds and urine samples, which exhibited an MIC of ≤8 µg/mL, whereas MIC of free antibiotics ranged from 32 to >1024 µg/mL. The results clearly indicate that the liposomes composed of naturally occurring oleic acid, could be used therapeutically either alone or in combination with antibiotics to effectively treat P. aeruginosa infections.

Novel pyochelin-based PEGylated liposomes for enhanced delivery of antibiotics against resistant clinical isolates of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a problematic human pathogen resistant to almost all available antibiotics. The important prerequisite for these drugs to target this bacterium is an efficient delivery system. Siderophore-mediated drug delivery system is a promising approach to carry out antibiotics to the cells. Pyochelin, a siderophore of P. aeruginosa, was successfully synthesized in a five-step procedure. PEGylated liposomal pyochelin-antibiotic (L-Pch-Ab) carrier was fabricated by thin-film hydration method. L-Pch-Ab had an average size of 90.31 ± 0.11 nm holding a negative zeta potential at -54.12 ± 0.03 mV (PDI <2). The MIC determined by broth dilution method against three clinical strains isolated from burn wounds showed that L-Pch-Ab significantly reduced (≤16 µg/ml) the MIC values than those of free antibiotics. In the time kill assay, L-Pch-Ab was bactericidal against all strains at most time intervals at 2 × and 4 × MIC up to 24 h. TEM observations revealed that L-Pch-Ab was actively taken up by P. aeruginosa and exhibited membrane deformation within 2 h. Developed L-Pch-Ab fused intimately with the outer membrane of MDRPa and exhibited effective antibacterial activity than free Ab. Furthermore, L-Pch-Ab kills MDRPa within infected HaCaT keratinocytes without any cytotoxic effects at 4× MIC concentrations after 72 h. Thus, the specific targeting of L-Pch-Ab with its higher efficacy to deliver drug by limiting the toxicity will be a novel approach to fight infections caused by P. aeruginosa.

Detection of blaCTX-M Extended Spectrum Beta-lactamase Producing Salmonella enterica Serotype Typhi in a Tertiary Care Centre.

INTRODUCTION: Infections caused by Salmonella are an important public health threat in tropical and subtropical countries. Due to the emergence of resistance to ampicillin, chloramphenicol and trimethoprim/sulfamethoxazole (multidrug resistant salmonellae) in the late 1980s, fluoroquinolones and extended spectrum cephalosporins became the drugs of choice. Resistance to cefotaxime and ceftriaxone due to the production of Extended Spectrum Beta-Lactamase (ESBL) and reduced susceptibility to ciprofloxacin have emerged resulting in treatment failure. The Cefotaximase (CTX-M) type ESBLs are the most widespread beta lactamase among Enterobacteriaceae including salmonellae. AIM: To detect the presence of blaCTX-M in salmonellae causing human infections. Detection of qnr genes to identify the coexistence of blaCTX-M and qnr gene. MATERIALS AND METHODS: The study included 103 consecutive, non-repetitive salmonellae isolated from clinical specimens obtained from July 2015- June 2016 which were identified up to species level by conventional/automated methods. Susceptibility to various classes of antimicrobial agents was determined by disc diffusion method. Minimum Inhibitory Concentration (MIC) to cefotaxime and ceftriaxone was determined by agar dilution method. The results were interpreted in accordance with Clinical & Laboratory Standard Institute (CLSI) (guidelines 2015. Detection of the ESBL phenotype was performed by the combined disk method. Polymerase Chain Reaction (PCR) amplification of all isolates was performed using group specific primers to characterize the presence of blaCTX-M, qnrA, qnrB and qnrS. RESULT: Of the 103 study isolates two isolates of Salmonella typhi were resistant to cefotaxime and ceftriaxone and had a MIC of 128µg/ml. PCR amplification and sequencing detected the presence of blaCTX-M-15 in these two isolates. These two isolates exhibited resistance to ciprofloxacin in vitro but qnr gene was not detected in these isolates. CONCLUSION: Resistance to third generation cephalosporins among salmonellae is a cause for concern as it may lead to treatment failure. It is imperative to continuously monitor the susceptibility pattern as enteric fever is endemic in India.

Carbapenemase-producing Enterobacteriaceae: Risk factors for infection and impact of resistance on outcomes.

BACKGROUND: Carbapenemase-producing Enterobacteriaceae (CPE) have increased in recent years leading to limitations of treatment options. The present study was undertaken to detect CPE, risk factors for acquiring them and their impact on clinical outcomes. METHODS: This retrospective observational study included 111 clinically significant Enterobacteriaceae resistant to cephalosporins subclass III and exhibiting a positive modified Hodge test. Screening for carbapenemase production was done by phenotypic methods, and polymerase chain reaction was performed to detect genes encoding them. Retrospectively, the medical records of the patients were perused to assess risk factors for infections with CPE and their impact. The data collected were duration of hospital stay, Intensive Care Unit (ICU) stay, use of invasive devices, mechanical ventilation, the presence of comorbidities, and antimicrobial therapy. The outcome was followed up. Univariate and multivariate analysis of the data were performed using SPSS software. RESULTS: Carbapenemase-encoding genes were detected in 67 isolates. The genes detected were New Delhi metallo-ß-lactamase, Verona integron-encoded metallo-ß-lactamase, and oxacillinase-181.Although univariate analysis identified risk factors associated with acquiring CPE infections as ICU stay (P = 0.021), mechanical ventilation (P = 0.013), indwelling device (P = 0.011), diabetes mellitus (P = 0.036), usage of multiple antimicrobial agents (P = 0.007), administration of carbapenems (P = 0.042), presence of focal infection or sepsis (P = 0.013), and surgical interventions (P = 0.016), multivariate analysis revealed that all these factors were insignificant. Mortality rate was 56.7% in patients with CPE infections. By both univariate and multivariate analysis of impact of the variables on mortality in these patients, the significant factors were mechanical ventilation (odds ratio [OR]: 0.141, 95% confidence interval [CI]: 0.024-0.812) and presence of indwelling invasive device (OR: 8.034; 95% CI: 2.060-31.335). CONCLUSION: In this study, no specific factor was identified as an independent risk for acquisition of CPE infection. However, as it is evident by multivariate analysis, there is an increased risk of mortality in patients with CPE infections when they are ventilated and are supported by indwelling devices.

Carriage of Multidrug Resistant Bacteria on Frequently Contacted Surfaces and Hands of Health Care Workers.

INTRODUCTION: Maximal contact between the patients and Health Care Workers (HCWs) happens in the Intensive Care Units (ICU). Control of nosocomial infections requires compliance with hand hygiene and contamination free surfaces. AIM: To determine the colonization of potential pathogens in the hands of HCWs and frequent contacted environmental surfaces. MATERIALS AND METHODS: A cross sectional study was conducted between September 2012 and May 2013 at Sri Ramachandra Medical College and Hospital. A total of 327 samples were collected using Glove juice technique from hands and swabs from frequently contacted surfaces. A sum of 157 samples were collected by glove juice technique from the hands of HCWs which included Consultants (20), Internees (3), Residents (10), Staff nurse (102) and support staff (22). A total of 170 samples were collected through swabbing which included frequently touched surfaces of apron and dress (140 which included 10 consultants, 3 internees, 9 Residents, 101 Staff nurse and 17 support staff), 9 door handle, 4 key board, 12 tap handles and 5 monitors. The samples were inoculated into Blood agar, Chocolate agar and Mac-Conkey agar plates and incubated at 37(0)C aerobically. The plates showing growth were further processed to identify the organisms by Gram staining and biochemical reactions. Antibiotic susceptibility testing was done for the isolates by Kirby-baur disc diffusion method as per CLSI guidelines. RESULTS: Out of the 157 hand sampling done by glove juice method 67(42.7%) of them showed growth and 90(57.3%) showed no growth. The potential pathogens grown were 13 (8.3%), consisting of Methicillin Sensitive Staphylococcus aureus (MSSA) 6(3.8%), Methicillin Resistant Staphylococcus aureus (MRSA) 2(1.3%), Pseudomonas spp 4(2.6%) and Acenitobacter spp 1 (0.6%). The MRSA was seen in Consultant 1(5%; n=20) and Staff nurse 1(0.9%; n= 102). Among the 140 sampling from the dress of HCWs growth was observed in 69(49.3%) and growth was absent in 71(50.7%). The potential pathogens observed were 14(10%) and they are MSSA 5(3.6%), MRSA 1 (0.7%), Pseudomonas spp 2(1.4%), Acenitobacter spp 3(2.1%) Enterobacter spp 1(0.7%), Klebseilla pneumoniae 1(0.7%) and Candida spp 1(0.7%). One MRSA was isolated from staff nurse (0.9%; n=101). Similarly multi-drug resistant Klebsiella pneumoniae 1(0.9%; n=102). Out of the 30 environmental samples 16(53.3%) showed growth and in 14(56.7%) growth was absent. The potential pathogens isolated were 3(10%) which included MSSA 2(6.6%) and MRSA 1(3.4%) and were isolated from the monitor. CONCLUSION: Adherence to infection control practices among all categories of HCWs is must for control of HAI. Glove juice method is a simple, easy and practical technique for determination of colonization of hands of HCWs and can be adapted as a methodology for screening the hands of HCWs.

Detection of Vancomycin Resistance among Enterococcus faecalis and Staphylococcus aureus.

INTRODUCTION: Vancomycin remains the drug of choice for resistant gram positive infections caused by Enterococcus spp and Methicillin resistant Staphylococcus aureus (MRSA). Increased use of vancomycin has led to frank resistance and increase in MIC (MIC creep). Vancomycin intermediate Staphylococcus aureus (VISA), Vancomycin resistant Staphylococcus aureus (VRSA) & Vancomycin resistant Enterococci (VRE) are important emerging nosocomial pathogens resulting in treatment failures. AIM: This study was undertaken to detect vancomycin resistance among clinical isolates of Staphylococcus aureus and Enterococcus faecalis by phenotypic and genotypic methods. MATERIALS AND METHODS: The study was conducted in a 1850 bedded university teaching hospital from November 2013 to April 2014. Non-repetitive, consecutive clinically significant Staphylococcus aureus (109) and Enterococcus faecalis (124) were included in this study. They were identified up to species level by conventional and automated methods. Susceptibility to various antibiotics was tested by disc diffusion method. MIC of vancomycin was determined by agar dilution method. Inducible resistance to clindamycin was detected by the D test. Methicillin resistance in Staphylococcus aureus (MRSA) was screened using cefoxitin disc. All isolates were subjected to polymerase chain reaction (PCR) to detect van A and van B genes. RESULTS: Out of 109 Staphylococcus aureus isolates, 54 were MRSA. By MIC there was no resistance observed to vancomycin.MIC50 was 1µg/ml. None of the isolates harboured van A and van B. Among Enterococcus faecalis, sixteen isolates (12.9%) and four isolates (3.2%) exhibited resistance to vancomycin and teicoplanin by disc diffusion respectively. All isolates were susceptible to linezolid. Van A was detected in 2, van B in 7 and one had both van A and van B. CONCLUSION: PCR remains the gold standard for diagnosis of vancomycin resistance. There was no resistance observed to vancomycin among Staphylococci though the MIC creep detected is a cause for concern. Eight percent of Enterococci were vancomycin resistant.