Role of ERG11 and MDR1 genes in cycloheximide and multidrug resistance in Candida species.

Candida species are amongst the commensals of the mucosal surfaces of the human body which include the oral cavity, vagina, and intestinal mucosa. Fungal infections are on the rise worldwide. The overall burden of infections due to fungi is difficult to estimate because the majority of them remain undiagnosed. The present study aims to determine the burden of antifungal resistance in low socioeconomic country, Pakistan and the frequency of ERG11 and MDR1 genes involved. A total of 636 Candida isolates were obtained from various tertiary care institutions in Lahore in the form of culture on various culture plates. Sabouraud agar culture plates were used to culture the Candida spp. Antifungal resistance was determined against Fluconazole, Itraconazole, Ketoconazole, and Nystatin via disk diffusion technique. Most resistance was observed against Fluconazole followed by Itraconazole, Ketoconazole, and Nystatin. The Candida isolates recovering from CVP tip and tissue have a high resistance profile. Candida species resistant to at least two antifungals were chosen for further ERG11 and MDR1 detection through real-time PCR. Among 255 Candida isolates, 240 contained ERG11 gene while MDR1 gene is present in 149 Candida isolates. The isolates carrying both genes were tested by the broth microdilution technique for the susceptibility against cycloheximide, all of them were able to grow in cycloheximide. The genetic determinants of antifungal resistance such as ERG11 and MDR1 are as important in the multidrug resistance against a variety of compounds and antifungal drugs.

Antimicrobial drug resistant features of Mycobacterium tuberculosis associated with treatment failure.

Tuberculosis stands as a prominent cause of mortality in developing countries. The treatment of tuberculosis involves a complex procedure requiring the administration of a panel of at least four antimicrobial drugs for the duration of six months. The occurrence of treatment failure after the completion of a standard treatment course presents a serious medical problem. The purpose of this study was to evaluate antimicrobial drug resistant features of Mycobacterium tuberculosis associated with treatment failure. Additionally, it aimed to evaluate the effectiveness of second line drugs such as amikacin, linezolid, moxifloxacin, and the efflux pump inhibitor verapamil against M. tuberculosis isolates associated with treatment failure. We monitored 1200 tuberculosis patients who visited TB centres in Lahore and found that 64 of them were not cured after six months of treatment. Among the M. tuberculosis isolates recovered from the sputum of these 64 patients, 46 (71.9%) isolates were simultaneously resistant to rifampicin and isoniazid (MDR), and 30 (46.9%) isolates were resistant to pyrazinamide, Resistance to amikacin was detected in 17 (26,5%) isolates whereas resistance to moxifloxacin and linezolid was detected in 1 (1.5%) and 2 (3.1%) isolates respectively. Among MDR isolates, the additional resistance to pyrazinamide, amikacin, and linezolid was detected in 15(23.4%), 4(2.6%) and 1(1.56%) isolates respectively. One isolate simultaneously resistant to rifampicin, isoniazid, amikacin, pyrazinamide, and linezolid was also identified. In our investigations, the most frequently mutated amino acid in the treatment failure group was Serine 315 in katG. Three novel mutations were detected at codons 99, 149 and 154 in pncA which were associated with pyrazinamide resistance. The effect of verapamil on the minimum inhibitory concentration of isoniazid and rifampicin was observed in drug susceptible isolates but not in drug resistant isolates. Rifampicin and isoniazid enhanced the transcription of the efflux pump gene rv1258 in drug susceptible isolates collected from the treatment failure patients. Our findings emphasize a high prevalence of MDR isolates linked primarily to drug exposure. Moreover, the use of amikacin as a second line drug may not be the most suitable choice in such cases.

Screening of C. auris among Candida isolates from various tertiary care institutions in Lahore by VITEK 2 and real time PCR based molecular technique.

Candida auris is a multidrug-resistant pathogen, that is a well-known cause of nosocomial infections. This pathogen is being identified using advanced diagnostic approaches and epidemiological typing procedures. In underdeveloped nations, several researchers developed and validated a low-cost approach for reliably identifying Candida auris. The goal of this study was to assess the burden of Candida auris in different teaching hospitals of Lahore and to limit its spread to minimize hospital-related illnesses. Candida isolates were obtained from various tertiary care institutions in Lahore in the form of culture on various culture plates. Sabouraud agar culture plates were used to culture the Candida spp. Fluconazole-resistant Candida species were chosen for further identification using VITEK 2 Compact ID and molecular identification using species-specific PCR assay. The current study obtained 636 Candida samples from several tertiary care institutions in Lahore. Fluconazole resistance was found in 248 (38.9%) of 636 Candida samples. No isolate was identified as Candida auris by VITEK 2 Compact ID and real-time PCR-based molecular identification. Thus with limited resources, these two methods may serve as useful screens for Candida auris. However, it should be screened all over the country to limit its spread to break the chain of nosocomial infections.

Molecular Characterization of Extensively Drug Resistant Salmonella Enterica Serovar Typhi Clinical Isolates from Lahore, Pakistan.

Background: The emergence of extensively drug-resistant (XDR) typhoid in Pakistan has endangered the treatment options available to manage this infection. Third generation cephalosporin were the empiric choice to treat typhoid fever in Pakistan, but acquisition of ESBLs have knocked them out of the arsenal. The current empiric choice is azithromycin which is vulnerable to resistance too. This study aimed to assess the burden of XDR typhoid and the frequency of resistance determinants in blood culture samples collected from different hospitals in Lahore, Pakistan. Methods: A total of 835 blood cultures were collected from different tertiary care hospitals in Lahore during January 2019 to December 2021. Among 835 blood cultures, 389 Salmonella Typhi were identified, and 150 were XDR S. Typhi (resistant to all recommended antibiotics). Antibiotics resistance genes of the first-line drugs (blaTEM-1, catA1, sul1, and dhfR7) and second line drugs (gyrB, gyrA, qnrS, ParC and ParE) were investigated among XDR S. Typhi. There were different CTX-M genes isolated using the specific primers, blaCTX-M-U, blaCTX-M-1, blaCTX-M-15, blaCTX-M-2, blaCTX-M-8 and blaCTX-M-9. Results: Antibiotic resistant genes of the first-line drugs were isolated with different frequency, blaTEM-1 (72.6%), catA1 (86.6%), sul1 (70%), and dhfR7 (56%). Antibiotics resistance genes of second-line drugs were isolated as: gyrB (60%), gyrA (49.3%), qnrS (32.6%), parC (44%) and parE (28%). Among CTX-M genes, blaCTX-M-U (63.3%) was the most frequent followed by blaCTX-M-15 (39.3%) and blaCTX-M-1 (26%). Conclusion: Our study concluded that XDR isolates circulating in Pakistan have acquired first-line and second-line antibiotic resistant genes quite successfully along with CTX-M genes (ESBLs) rendering them resistant to the third generation cephalosporins as well. Emergence of azithromycin resistance in XDR S. Typhi which is currently used as an empiric treatment option is worrisome and needs to be monitored carefully in endemic countries like Pakistan.

Antibacterial assessment of commercially available hand sanitizers in Pakistan by EN-1500.

BACKGROUND: The effectiveness of hand sanitizers marketed to the general population is essential for infection prevention and control. Main theme of the study was that whether the commercially available hand sanitizers meet the WHO recommended standards in terms of efficacy? Current study aims to investigate the efficacy of ten commercially available hand sanitizers. METHODS: The methodology was based on European Standard EN-1500. Following the artificial contamination of hands, pre and post samples were obtained to determine the log reduction values for each sanitizer. RESULTS: The results showed that out of ten only one sanitizer showed highest log reduction which was comparable to the reference product. Product B was most efficient in sanitization of hands with mean log reduction of 6.00 ± 0.15. The lowest sanitization efficacy was recorded for product F with mean log reduction of 2.40 ± 0.51, however the reference product 2-propanol result in mean log reduction of 6.0 ± 0.00. The products used in this study show a statistical significant results (p value: < 0.01). CONCLUSION: It is concluded that only one product showed active sanitizer efficacy. This study provides an important insight for manufacturing company and authorizing authorities to assess the efficacy of hand sanitizer. Hand sanitization is one approach to stop the spread of diseases carried on by harmful bacteria inhabiting our hands. Apart from the manufacturing strategies, ensuring proper use and quantity of hand sanitizers is very important.

Reporting of Azithromycin Activity against Clinical Isolates of Extensively Drug-Resistant Salmonella enterica Serovar Typhi.

This study aimed to evaluate the minimum inhibitory concentration (MIC) of azithromycin (AZM) in clinical isolates of extensively drug-resistant (XDR) Salmonella Typhi (i.e., resistant to chloramphenicol, ampicillin, trimethoprim-sulfamethoxazole, fluoroquinolones, and third-generation cephalosporin) using the E-test versus the broth microdilution method (BMD). From January to June 2021, a retrospective cross-sectional study was carried out in Lahore, Pakistan. Antimicrobial susceptibility was performed initially by the Kirby-Bauer disk diffusion method for 150 XDR Salmonella enterica serovar Typhi isolates, and MICs of all the recommended antibiotics were determined by the VITEK 2 (BioMérieux) fully automated system using Clinical Laboratory Standard Institute (CLSI) 2021 guidelines. The E-test method was used to determine AZM MICs. These MICs were compared with the BMD, which is the method recommended by the CLSI but not adopted in routine laboratory reporting. Of 150 isolates, 10 (6.6%) were resistant by disk diffusion. Eight (5.3%) of these had high MICs against AZM by the E-test. Only three isolates (2%) were resistant by E-test, having an MIC of 32 µg/mL. All eight isolates had a high MIC by BMD with different MIC distributions, but only one was resistant, having an MIC of 32 µg/mL by BMD. The sensitivity, specificity, negative predictive value, positive predictive value, and diagnostic accuracy of the E-test method versus BMD were 98.65%,100%, 99.3%, 33.3%, and 98.6%, respectively. Similarly, the concordance rate was 98.6%, negative percent agreement was 100%, and positive percent agreement was 33%. The BMD is the most reliable approach for reporting AZM sensitivity in XDR S. Typhi compared with the E-test and disk diffusion methods. Potentially, AZM resistance in XDR S. Typhi is around the corner. Sensitivity patterns should be reported with MIC values, and if possible, higher values should be screened for the presence of any potential resistance genes. Antibiotic stewardship should be strictly implemented.

Emergence of multidrug-resistant ST11 blaKPC-2 producing Klebsiella pneumoniae coharboring blaCTX-M and blaSHV in Pakistan.

INTRODUCTION: Carbapenemases are primarily responsible for the intensified spread of multidrug-resistant (MDR) K. pneumoniae by virtue of antibiotics overuse. Therefore, frequent investigation of high-risk clones especially from developing world is crucial to curtail global spread. METHODOLOGY: In this observational study, 107 K. pneumoniae were retrieved and confirmed genotypically from April 2018 to March 2020 from tertiary care hospitals in Lahore, Pakistan. Carbapenemases and extended-spectrum ß-lactamases were verified by Polymerase Chain Reaction and Sanger sequencing. Multilocus sequence typing and plasmid replicon typing were used to assign clonal lineages and plasmid replicons. RESULTS: Among the K. pneumoniae, 72.9% (78/107) strains were carbapenem resistant (CR) with 65.4% (51/78) exhibiting carbapenemase producing phenotype. Among CR K. pneumoniae 38.5% (30/78) strains exhibited the following carbapenemase genotypes: blaNDM-1 (26.7%, 8/30), blaOXA-48 (26.7%, 8/30), blaKPC-2 (20.0%, 6/30), blaVIM (10.0%, 3/30), blaNDM-1/blaOXA-48 (10.0%, 3/30), blaOXA-48/blaVIM (3.3%, 1/30) and blaOXA-48/blaIMP (3.3%, 1/30). Tigecycline and polymyxin-B retained susceptible profile. ß-lactam drugs showed intermediate to high resistance. The occurrence of CR K. pneumoniae infections was significantly associated with wound (39.7%, p = 0.0007), pus (38.5%, p = 0.009), general surgery (34.6%, p = 0.002) and intensive-care unit (26.9%, p = 0.04). blaKPC-2 producing K. pneumoniae coharboring blaCTX-M/blaSHV (66.7%) and blaCTX-M (33.3%) exhibited sequence type (ST) 258 (n = 4) and ST11 (n = 2) sequence types with IncFII, IncN, IncFIIA, IncL/M and IncFIIK plasmids. CONCLUSIONS: This is the first report describing the emergence of MDR blaKPC-2 producing K. pneumoniae ST11 coharboring blaCTX-M and blaSHV in Pakistan.

Identifying and elucidating the resistance of Staphylococcus aureus isolated from hospital environment to conventional disinfectants.

BACKGROUND: Staphylococcus aureus is a nosocomial pathogen, detection and elucidation of its resistance mechanisms to conventional disinfectants may aid in limiting its spread on environmental surfaces in health care settings. In the current study, disinfectant susceptibility of S. aureus strains isolated from the hospital environment as well as possible associations between the presence of disinfectant-resistance genes and reduced susceptibility to disinfectants was investigated. METHODS: A total of 245 samples were collected from the hospital environmental surfaces. The minimum inhibitory (MIC) and bactericidal concentrations (MBC) of disinfectants against S. aureus isolates were determined using the micro-broth dilution method. The qac genes (qacA, qacE, and qacΔE1) were detected by PCR and confirmed by sanger sequencing. RESULTS: A total of 47 S. aureus strains were isolated, with more than 85% of them showing methicillin resistance. The qacA, qacE, and qac∆E1 genes were found in 23.4%, 29.7%, and 4.2% isolates respectively. All the isolates with qac genes had higher MIC and MBC values to selected disinfectants. CONCLUSIONS: Significant methicillin resistant S. aureus (MRSA) contamination in the hospital environment was detected. Furthermore, higher qac gene frequencies were found in MRSA isolates that also correlated with higher MIC/MBC values to different disinfectants. The study proposes that hospitals should develop policies to determine disinfectant MICs against the common environmental isolates to contain the spread of resistant strains.

Investigation of Three Morchella Species for Anticancer Activity Against Colon Cancer Cell Lines by UPLC-MS-Based Chemical Analysis.

In search of new anticancer agents, natural products including fungal compounds had been used as potential anticancer agents. The aim of this study was to investigate the anticancer activity of Morchella extracts against colon cancer cell line and UPLC-DAD-MS/MS analysis for the identification of compounds. The cytotoxic activity of the three Morchella species was examined for their anti-carcinogenic properties against the colon cancer cell lines. Phytochemical analyses were performed to screen Morchella for the presence of anti-cancerous compounds. All the fungal extracts inhibited the viability of colon cancer cells in a dose-dependent manner. Major compounds identified in Morchella included amino acid, fatty acid, sterol, flavonoid, peptide, glutamic acid, alkaloid, terpenoid, cyclopyrrolones, and coumarin. Several new compounds were detected among all the three Morchella extracts. In conclusion, all the fungal extracts showed potential inhibition of colon cancer cells and actively arrested the cell viability. It was concluded that the identified bioactive compounds might be the main constituents contributing to the anticancer activity of Morchella against human colon cancer cell lines. Thus, Morchella extracts are a potential source of bioactive compounds with cytotoxicity and could potentially be used as functional food supplements. Due to the nature of impressive findings, this investigation should be undertaken further to allow the studies to explore and develop a potential cytotoxic agents against colon cancer.

Emergence of high colistin resistance in carbapenem resistant Acinetobacter baumannii in Pakistan and its potential management through immunomodulatory effect of an extract from Saussurea lappa.

Carbapenem resistant Acinetobacter baumannii has emerged as one of the most difficult to treat nosocomial bacterial infections in recent years. It was one of the major causes of secondary infections in Covid-19 patients in developing countries. The polycationic polypeptide antibiotic colistin is used as a last resort drug to treat carbapenem resistant A. baumannii infections. Therefore, resistance to colistin is considered as a serious medical threat. The purpose of this study was to assess the current status of colistin resistance in Pakistan, a country where carbapenem resistant A. bumannii infections are endemic, to understand the impact of colistin resistance on virulence in mice and to assess alternative strategies to treat such infections. Out of 150 isolates collected from five hospitals in Pakistan during 2019-20, 84% were carbapenem resistant and 7.3% were additionally resistant to colistin. There were two isolates resistant to all tested antibiotics and 83% of colistin resistant isolates were susceptible to only tetracycline family drugs doxycycline and minocycline. Doxycycline exhibited a synergetic bactericidal effect with colistin even in colistin resistant isolates. Exposure of A. baumannii 17978 to sub inhibitory concentrations of colistin identified novel point mutations associated with colistin resistance. Colistin tolerance acquired independent of mutations in lpxA, lpxB, lpxC, lpxD, and pmrAB supressed the proinflammatory immune response in epithelial cells and the virulence in a mouse infection model. Moreover, the oral administration of water extract of Saussuria lappa, although not showing antimicrobial activity against A. baumannii in vitro, lowered the number of colonizing bacteria in liver, spleen and lung of the mouse model and also lowered the levels of neutrophils and interleukin 8 in mice. Our findings suggest that the S. lappa extract exhibits an immunomodulatory effect with potential to reduce and cure systemic infections by both opaque and translucent colony variants of A. baumannii.

Staphylococcal Cassette Chromosome mec Typing and Multilocus Variable Number Tandem Repeat Analysis of Methicillin Resistant Staphylococcus aureus Clinical Isolates with Vancomycin Creep Phenomenon.

Background: The association of treatment failure and mortality with vancomycin minimum inhibitory concentration creep (MIC) is a matter of serious concern in patients with severe methicillin resistant Staphylococcus aureus (MRSA) infections. The purpose of the study was to identify and characterize staphylococcal cassette chromosome mec (SCCmec) and clonal types of MRSA strains, exhibiting the vancomycin MIC creep phenomenon. Methods: A total of 3305 S. aureus strains were isolated from various clinical samples of Lahore General Hospital, Lahore, Pakistan. MRSA strains were identified by cefoxitin resistant (≤21mm) followed by mecA and mecC gene genotyping. Vancomycin MIC creep was determined by E-test. Isolates having MIC values >1.5 µg/mL were further subjected for SCCmec typing (I-V and XI) and multiple-locus variable number tandem repeat analysis (MLVA) by amplification of spa, sspA, clfA, clfB, and sdrCDE genes. A dendrogram was created based on the similarity index using bioneumerics software. Results: About 13.3% (440/3305) isolates were MRSA with 99.3% (437/440) and 0.7% (3/440) carried mecA and mecC genes, respectively. In 120 MRSA isolates, the MIC of vancomycin was >1.5µg/mL. In MRSA isolates with high vancomycin MIC (>1.5µg/mL), the most common SCCmec type was SCCmec III (38.3%), followed by SCCmec IVa (15.8%), SCCmec IIIa (13.3%,), SCCmec IVc (7.5%), SCCmec IVe (5.8%), SCCmec IVd (5.8%), SCCmec IVb (4.2%), SCCmec II (2.5%), SCCmec V (1.7%), SCCmec I (1.7%) and SCCmec XI (1.7%). MLVA revealed 60 genotypic groups of MRSA isolates having a 92% similarity index. Conclusion: SCCmec III was the most common type in genetically related MRSA isolates showing vancomycin MIC creep. The presence of SCCmec XI may further add burden to infection control measures.

Antibacterial Activity of Different Extracts of Ascomata of Morchella conica and M. esculenta (Ascomycota) against Salmonella Species.

Antibiotic resistance has become a worldwide concern, as it has reduced the clinical efficiency of multiple antibiotics. As a result, screening of novel antibacterial substances for antimicrobial potential has increased. Mushrooms are widely known as a source of antimicrobial agents. The current study was designed to investigate the bacteriostatic and bactericidal effects of Morchella conica and M. esculenta against typhoidal and nontyphoidal Salmonella species. The sensitivity of S. typhi, S. paratyphi-A, and S. typhimurium was determined using an agar diffusion assay. The standard broth microdilution method was used to assess the minimal inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC). Bacterial growth and inhibition kinetics were evaluated spectrophotometrically. All of the mushroom extracts were able to inhibit the growth of typhoidal and nontyphoidal Salmonella species. Notably, all of the extracts possessed bacteriostatic effects (MIC: 3.33 ± 0.6 to 16.0 ± 0 mg/mL) and bactericidal effects (MBC: 8-16 mg/mL). The results showed statistically significant differences of antibacterial and bactericidal potential of mushroom extracts against the tested bacteria (P ≤ 0.05). Thus, extracts of Morchella species can be used as natural antibacterial pharmaceuticals. Further mycopharmacological studies must be performed to characterize their metabolites.

Investigation of Morchella esculenta and Morchella conica for their antibacterial potential against methicillin-susceptible Staphylococcus aureus, methicillin-resistant Staphylococcus aureus and Streptococcus pyogenes.

Antimicrobial resistance is an alarming problem, especially due to emergence of methicillin-resistance Staphylococcus aureus (MRSA). World Health Organization (WHO) has already listed MRSA as a top priority pathogen for the development of novel antibacterial agents. Presently, different therapeutic approaches against bacterial infections are in practice which includes targeting bacterial virulence factors, bacteriophage therapy, and manipulation of the microbiome. Natural products have been efficiently used for centuries to combat bacterial infections. Morchella is a natural fungal product which has been reported to possess broad-spectrum biological activities against bacterial infections. Hence, this study was aimed to evaluate the antibacterial efficacy of two macro-fungi against S. aureus, MRSA, and Streptococcus pyogenes (S. pyogenes). The antibacterial potential of both fungal extracts (Morchella esculenta and Morchella conica) was evaluated using disk diffusion and standard broth microdilution methods. The chemical compounds of both fungi were investigated using ultra-performance liquid chromatography mass spectroscopy (UPLC-MS) analysis. All fungal extracts inhibited growth of tested bacteria with inhibitory zone ranging from 10.66 ± 0.3 to 21.00 ± 1.5 mm. The minimum inhibitory concentration (MIC) of tested bacterial growth ranged from 03.33 to 16.0 mg/ml. It was noteworthy that Morchella extracts prevented S. aureus growth in a bactericidal manner with minimal bactericidal concentration (MBC) of 8-16 mg/ml. The extracts were also more effective against MRSA than currently available antibiotics. In conclusion, the growth inhibition of tested bacteria by fungal extracts revealed their potential as antibacterial agents and their compounds may be used as drug candidates.

Four year trend of antimicrobial susceptibility of methicillin-resistant Staphylococcus aureus in a tertiary care hospital, Lahore.

OBJECTIVE: To determine the susceptibility pattern of methicillin-resistant staphylococcus aureus to different antibiotics. METHODS: The descriptive study was conducted at the Microbiology Department of the University of Health Sciences, Lahore, Pakistan, from August 2016 to July 2019, and comprised staphylococcus aureus samples that were processed and identified using colony morphology on blood agar, gram stain, catalase, coagulase and deoxyribonuclease testing. Screening for methicillin-resistant staphylococcus aureus was done using cefoxitin disc 30µg and oxacillin disc 1mg. Antimicrobial susceptibility was tested using the modified Kirby-Bauer disc diffusion method in line with the Clinical and Laboratory Standards Institute guidelines 2019. Data was analysed using SPSS 24. RESULTS: Of the 2704 strains processed, 402(14.86%) were found to be methicillin-resistant staphylococcus aureus. Of them, 204(50.74%) were recovered from pus, while 10(2.48%) were recovered from urine. All 402(100%) isolates were sensitive to vancomycin and linezolid, and resistant to penicillin, followed by erythromycin 306(76.11%) and sulfamethoxazole/ trimethoprim 295(73.38%). Overall, lower resistance was seen with doxycycline 145(36.06%) and clindamycin 160(39.80%). Inducible clindamycin resistance was seen in 142(35.23%) isolates. CONCLUSIONS: An efficacious susceptibility pattern of methicillin-resistant staphylococcus aureus was seen with vancomycin and linezolid, moderate susceptibility with doxycycline and clindamycin, while high resistance was observed for penicillin, erythromycin and trimethoprim/sulfamethoxazole.

From Molecular Pathology of COVID 19 to Nigella Sativum as a Treatment Option: Scientific Based Evidence of Its Myth or Reality.

COVID-19 virus is a causative agent of viral pandemic in human beings which specifically targets respiratory system of humans and causes viral pneumonia. This unusual viral pneumonia is rapidly spreading to all parts of the world, currently affecting about 105 million people with 2.3 million deaths. Current review described history, genomic characteristics, replication, and pathogenesis of COVID-19 with special emphasis on Nigella sativum (N. sativum) as a treatment option. N. sativum seeds are historically and religiously used over the centuries, both for prevention and treatment of different diseases. This review summarizes the potential role of N. sativum seeds against COVID-19 infection at levels of in silico, cell lines and animal models.

Molecular detection of blaOXA-23 gene and blaOXA-51 gene in carbapenem resistant strains of Acinetobacter baumannii in patients with ventilator associated pneumonia at tertiary care hospitals.

OBJECTIVE: To evaluate carbapenem resistance and to detect blaOXA-23 and blaOXA-51 genes in carbapenem-resistant Acinetobacter baumanii isolates recovered from patients having pneumonia secondary to ventilation. METHODS: The cross-sectional study was conducted from July 2017 to June 2018 at the Department of Microbiology, University of Health Sciences, Lahore, Pakistan, and comprised endotracheal aspirates / tracheobroncheal lavage samples from patients irrespective of age and gender who developed pneumonia after being on the ventilator for 48 hrs at the Combined Military Hospital, and Jinnah Hospital, Lahore. The samples were inoculated on MacConkey and blood agar and aerobically incubated at a temperature of 370C for 18-24 hours. The isolated organisms were further assessed by standard morphological, cultural and biochemical profile. Antibiotic susceptibility was done by Kirby-Bauer disc diffusion method. Carbapenem-resistant Acinetobacter baumannii were checked for carbapenemase production using Modified Hodge Test. Conventional polymerase chain reaction and agarose gel electrophoresis were performed to detect blaOXA-23 and blaOXA-51 genes. Data was analysed using SPSS 17. RESULTS: Out of 157 samples, 92(58.6%) yielded growth of bacteria, and, among them, 39(42.4%) were identified as Acinetobacter baumannii. All (100%) Acinetobacter baumannii cases showed resistance to carbapenem, were producing carbapenemase enzyme, and were positive for blaOXA-51 gene. The blaOXA-23 gene was amplified in 38(97.4%) isolates. CONCLUSIONS: BlaOXA-23 gene appeared to be the major cause of carbapenem resistance.

Clonal relatedness and plasmid profiling of extensively drug-resistant New Delhi metallo-ß-lactamase-producing Klebsiella pneumoniae clinical isolates.

Aim: Carbapenem-resistant Klebsiella pneumoniae (CR-KP) particularly New Delhi metallo-ß-lactamase (NDM) is a serious public health concern globally. The aim of the study to determine the molecular epidemiology of blaNDM-producing clinically isolated K. pneumoniae. Methods: Carbapenem-resistant K. pneumoniae isolates (n = 100) were collected from tertiary care hospital Lahore. Isolates were confirmed by VITEK® 2 system and MALDI-TOF. Minimum inhibitory concentration was performed by VITEK 2 and molecular characterization was done by PCR, PFGE, DNA hybridization and replicon typing. Results: Of 90 MBL-producing K. pneumoniae, 75 were NDM producers; 60 were NDM-1 and 11 NDM-5. A total of 27 K. pneumoniae belonged to ST11 and 14 to ST147. NDM-positive isolates were 100% resistant to ß-lactam antibiotics except for colistin. 13.3% isolates carried blaNDM on ∼140 kb plasmids. A total of 32 (52.4%) isolates were positive for IncA/C and 18 (29.5%) IncF/II. Conclusion: The extensively resistant lineage of NDM-producing K. pneumoniae is prevalent in the clinical setting.

Emergence of plasmid-mediated mcr genes from Gram-negative bacteria at the human-animal interface.

BACKGROUND: The global emergence of plasmid-mediated colistin resistance (Col-R) conferred by mcr genes in gram-negative rods (GNRs) has jeopardized the last treatment option for multidrug-resistant bacterial infections in humans. This study aimed to assess the emergence of mcr gene-mediated Col-R in GNRs isolated from humans and animals in Pakistan. METHODS: Animal and clinical specimens collected from various sources were prospectively analysed using standard microbiological procedures. Pathogens were identified using the API 20E and API 20NE systems (bioMerieux). Minimum inhibitory concentration (MIC) against colistin was determined using the MIC detection methods, and multiplex polymerase chain reaction (PCR) was used to amplify the mcr-1 to mcr-5 genes. RESULTS: We isolated 126 (88.1%) animal and 17 (11.9%) human Col-R phenotypes, among which there was a significant association (P < 0.01) of Escherichia coli and Proteus mirabilis with animals and of Acinetobacter baumannii with humans. Animal strains exhibited statistically significant (P < 0.05) resistance to co-trimoxazole, chloramphenicol, and moxifloxacin, and the human pathogens exhibited statistically significant (P < 0.05) antibiotic resistance to cephalosporins, carbapenems, and piperacillin-tazobactam. For Col-R strains, MIC50 values were > 6 µg/mL and > 12 µg/mL for human and animal isolates, respectively. mcr genes were detected in 110 (76.9%) bacterial strains, of which 108 (98.2%) were mcr-1 and 2 (1.8%) were mcr-2. CONCLUSIONS: The detection of a considerable number of mcr-1 and mcr-2 genes in animals is worrisome, as they are now being detected in clinical pathogens. The acquisition of mcr genes by colistin-susceptible bacteria could leave us in a post-antibiotic era.

Assessment of Vancomycin MIC Creep Phenomenon in Methicillin-Resistant Staphylococcus aureus isolates in a Tertiary Care Hospital of Lahore.

OBJECTIVE: To assess vancomycin MIC creep phenomenon in methicillin-resistant Staphylococcus aureus isolated from clinical specimens. METHODS: This descriptive study was conducted in Microbiology department of University of Health Sciences, Lahore from January 2016- December 2019. In this study, vancomycin MICs were revealed by E test method for clinical MRSA strains. For the final evaluation, a single isolate from each patient was taken. The reported vancomycin MICs results were used and the values were not rounded up to the next upward value. For every study year, MIC50, MIC90, median and geometrical mean MIC, percentages of susceptible and resistant strains were calculated. RESULTS: A total of 352 MRSA strains were isolated out of 2704 staphylococcal isolates. Our study showed elevated vancomycin MIC among MRSA isolates. The majority of isolates showed MIC values ≥1.5µg/ml. MIC50, MIC 90 was constant throughout four years period. However, geometric mean MIC increased gradually during the study period. The MIC greater than base year median was overall 17.3%. A complete shift can be observed between MIC "1.0" and "2.0" the percent of cases with MIC "1.0" decreased and with MIC "2.0" increased over time crossing each other in 2017. CONCLUSION: Vancomycin MIC creep was identified in clinical isolates of MRSA, during four years of study period. Even though there is an absence of VISA and VRSA strains; this significant increase in vancomycin MIC trend is indeed worrying for the clinicians about the threat of potential failure of treatment in MRSA infections.

High prevalence of carbapenem-resistant Acinetobacter baumannii associated respiratory tract infections in Pakistani hospitals.

Acinetobacter baumannii is one of the most common causes of nosocomial infections in developing countries. It has a better ability to get antimicrobial resistance due to plasticity in genome. The recent emergence of carbapenem-resistant A. baumannii isolates in several countries narrows down the spectrum of options to treat A. baumannii infections. The WHO has placed carbapenem-resistant A. baumannii on the top of priority organisms against which novel antibiotics are required. A systematic evaluation of carbapenem-resistant A. baumannii infections in three tertiary care hospitals in Pakistan is presented here. A total of 2270 positive culture samples were collected over a period of two years. Of which 1642 (72.33%) were respiratory tract specimens. A. baumannii was identified in 681 (41 %) cases. Of which 583 (85.5%) were carbapenem-resistant. Our findings suggest that the burden of carbapenem-resistant A. baumannii infections is alarmingly high in Pakistan.