Detection of high-risk human papillomavirus infected cervical biopsies samples by immunohistochemical expression of the p16 tumor marker.

Cervical cancer is the fourth most common type of cancer in women worldwide. It is widely accepted that the main cause of cervical cancer, especially in underdeveloped countries like Pakistan, is the infection caused by the human papillomavirus (HPV). The current screening and diagnostic methods face several challenges in accurately detecting the various types of lesions caused by HPV. Therefore, the present study was conducted to assess the effectiveness of p16 immunohistochemistry (IHC) analysis as a diagnostic method in samples of cervical biopsies. One hundred cervical biopsy samples were obtained from female patients across various age groups (> 20- ≤ 30, > 31- ≤ 40, > 41- ≤ 50, > 51- ≤ 60 years). These samples were subsequently prepared for subsequent examination. All samples were analyzed using automated tissue processing followed by Hematoxylin and Eosin (H & E) staining, and p16 IHC tumour marker staining. The H & E slides showed changes in normal cervical tissues, while four cervical abnormalities were identified statistically significant using p16 marker including chronic cervicitis, nabothian cyst formation, cervical intraepithelial neoplasia, and cervical cancers (P value 0.014). Furthermore, among females of different age groups (> 31- ≤ 40, > 41- ≤ 50, > 51- ≤ 60 years) were found statistically significant suffering from cervical cancer (P value 0.04), HPV with cervical cancer (P value 0.01), HPV with cervical intraepithelial neoplasia (P value 0.01). Based on the available data, it can be inferred that the incorporation of the p16 tumor marker may be a valuable method for detecting high-risk HPV in cervical biopsies samples.

Transcriptional Response of Multidrug-Resistant Klebsiella pneumoniae Clinical Isolates to Ciprofloxacin Stress.

BACKGROUND: The term "persisters" refers to a small bacterial population that persists during treatment with high antibiotic concentration or dose in the absence of genetic resistance. The present study was designed to investigate the transcriptional response in indigenous Klebsiella pneumoniae under the ciprofloxacin stress. METHODS: Isolation and identification of K. pneumoniae were carried out through standard microbiological protocols. The characterization of quinolone resistance was performed by estimating the quinolone susceptibility testing, MIC estimation, and detecting the QRDR and PMQR. Transcriptional response of the isolates to ciprofloxacin was determined using qPCR. RESULTS: Among 34 isolates, 23 (67%) were resistant to ciprofloxacin. Both QRDR (gyrA and gyrB) and PMQR (qnrA, qnrB, and qnrS) were detected in the isolates, and all were found resistant to ciprofloxacin. The mRNA levels of both mutS and euTu under the influence of ciprofloxacin were significantly increased. On ciprofloxacin exposure, the mRNA levels of the DNA damage response element (mutS) were raised in a time-dependent fashion. K. pneumoniae showed high-level resistance to ciprofloxacin in the presence of mutations in QRDR and PMQR genes. CONCLUSION: The transcriptional response revealed the upregulation of DNA repair and protein folding elements (mutS and euTu) in ciprofloxacin stress and delayed cell division. The ciprofloxacin was found to trigger various stress responses in a time- and concentration-dependent manner.

Antibacterial efficacy of silver nanoparticles against metallo-ß-lactamase (blaNDM, blaVIM, blaOXA) producing clinically isolated Pseudomonas aeruginosa.

Carbapenem resistance in Pseudomonas aeruginosa is a major concern in the public health sector, primarily in developing countries such as Pakistan. Therefore, novel approaches such as Silver nanoparticles (AgNPs) can be used to address emerging concerns. Clinical isolates (n=200) were reconfirmed using selective media and API 20NE kit. The antibiogram was determined according to the CLSI 2016 guidelines. Molecular detection was carried out by PCR. Antibacterial activity in AgNPs was achieved by dilution method. Of 200 P. aeruginosa, mostly (n=82; 41%) were isolated from pus samples. Of 110 MDR P. aeruginosa, 70 (63%) were carbapenemase and 58 (52%) were MBL producers. Antimicrobial profile of MBL producing P. aeruginosa reported that all isolates were resistant to ß-lactams, and 89% to levofloxacin and ciprofloxacin except colistin. Of 25 (35.7%) blaNDM producing P. aeruginosa, 12 isolates (48%) had MIC 16µg/mL to imipenem. Of 23 (32%) blaVIM producing P. aeruginosa, 12 (52%) contained MIC 16µg/mL to imipenem. However, 12 (17.1%) blaOXA-48 producing P. aeruginosa, 4 (33%) contained MIC 16µg/mL to imipenem. In vitro AgNPs activity inhibited and killed MBL producing isolates at 1 mg/mL and 2 mg/mL, respectively. AgNPs may be used as an alternative therapy followed by multiple clinical trials.

Gut Microbiota and Metabolic Disorders: Advances in Therapeutic Interventions.

Human gut microbiota consist of numerous microorganisms, but the most abundant species are Bacteroides and Firmicutes. Each human possesses a specific gut microbiota, which can be altered by diet, antibiotics, lifestyle, and genetic background. Gut microbiota perform vital functions, but in this article, we aimed to elaborate the effects of modified composition of microbiota on host metabolism. Ligands for G protein coupled receptors (GPCRs) are short-chain fatty acids (SCFAs) located on endocrine glands, epithelial cells, and adipocytes. SCFAs are produced in the distal gut by bacterial fermentation of nondigestible polysaccharides; they induce the various beneficial effects including decrease serum glucose level, insulin resistance, as well as inflammation; and they increase glucagon-like peptide-1 (GLP-1) secretion. Fasting-induced adipose factor (FIAF) is suppressed by gut microbiota and results in the increased storage of fatty acids in the adipose tissues and liver. An increased lipopolysaccharide level due to altered gut microflora cause the initiation of inflammation associated with type 2 diabetes mellitus (T2DM). Intestinal dysbiosis and metabolic endotoxemia are considered key mechanisms that seem to be associated with the development of T2DM and obesity. Therapeutic interventions that can be used for the treatment of diabetes include metformin, dietary modulation, probiotics, prebiotics, fecal microbiota transplantation and bariatric surgery.

Frequency of multi drug resistant Pseudomonas aeruginosa in different wound types of hospitalized patients.

Pseudomonas aeruginosa colonization is one of the major complications of wound infection leading to higher risk of morbidity and mortality. The trend of antibiotic resistant against Pseudomonas aeruginosa is increasing day by day due to irregular and extensive use of antibiotics. The main aim of this cross- sectional study is to detect the frequency of MDR Pseudomonas aeruginosa among various types of wounds during January to December 2018. In this study total 532 clinical samples were collected from wounded patients and subjected to the isolation and identification of Pseudomonas aeruginosa by standard microbiological techniques. Molecular identification of the isolates was done through PCR by using specific primers against Oprl, OprL and PA-SS genes of Pseudomonas aeruginosa. Antibiotic susceptibility testing and minimum inhibitory concentration was done by disc diffusion method and broth dilution assay respectively. PCR was performed for the molecular detection of ESBL and MBL genes using specific primers. Out of total 532 clinical samples 203 (38%) samples were identified as Pseudomonas aeruginosa. Out of positive samples 119 (58.6%) were confirmed MDR Pseudomonas aeruginosa. Out of 119 MDR positive samples, burn wounds showed the highest percentage 43 (36%), while least percentage 4 (3%) of MDR Pseudomonas aeruginosa was found in surgical wounds (P<0.05). All the selected isolates were resistant to ß-lactams drugs and most effective drugs were tigecycline and colistin. Highest prevalence in the infected wound patients is blaNDM 14 (25.9%) producing P. aeruginosa and least blaKPC 1 (1.8%) producing P. aeruginosa. Results of the study concluded that surgical wounds showed the highest prevalence of MDR P. aeruginosa, suitable measures should be adopted to restrain this public health menace.

In vitro antibiofilm and anti-adhesion effects of magnesium oxide nanoparticles against antibiotic resistant bacteria.

The aim of the current investigation was to determine the antibacterial and antibiofilm potential of MgO nanoparticles (NPs) against antibiotic-resistant clinical strains of bacteria. MgO NPs were synthesized by a wet chemical method and further characterized by scanning electron microscopy and energy dispersive X-ray. Antibacterial activity was determined by broth microdilution and agar diffusion methods. The Bradford method was used to assess cellular protein leakage as a result of loss of membrane integrity. Microtiter plate assay following crystal violet staining was employed to determine the effect of MgO NPs on biofilm formation and removal of established biofilms. MIC values ranged between 125 and 500 µg/mL. Moreover, treatment with MgO NPs accelerated rate of membrane disruption, measured as a function of leakage of cellular proteins. Leakage of cellular protein content was greater among gram-negative bacteria. Cell adherence assay indicated 25.3-49.8% inhibition of bacterial attachment to plastic surfaces. According to a static biofilm method, MgO NPs reduced biofilm formation potential from 31% to 82.9% in a time-dependent manner. Moreover, NPs also significantly reduced the biomass of 48, 72, 96 and 120 hr old biofilms (P < 0.05). Cytotoxicity experiments using a neutral red assay revealed that MgO NPs are non-toxic to HeLa cells at concentrations of 15-120 µg/mL. These data provide in vitro scientific evidence that MgO NPs are effective and safe antibiofilm agents that inhibit adhesion, biofilm formation and removal of established biofilms of multidrug-resistant bacteria.

Sero-epidemiology and Risk Factor Analysis of Measles Among Children in Pakistan.

Comparative cross sectional study was conducted on blood samples (n = 231) collected from children of 1 to 10 years of age in Punjab Pakistan through convenient sampling method. Indirect haemagglutination assay (IHA) was standardized and used for serodiagnosis and evaluation of humoral immunity against measles. Associated risk factors including age, gender, locale, and vaccination status were analyzed. Geometric mean titre (GMT) of vaccinated individuals was significantly higher (p < 0.001) than that of non-vaccinated individuals showing that IHA titre of vaccinated individuals was a measure of humoral immune response; whereas, in case of non-vaccinated individuals an indicative of exposure to the measles infection.

Role of chemical and herbal dentifrices against indigenous oral Pathogens.

The oral cavity has its own significant micro-flora but under unhygienic conditions can cause infections or diseases like gingivitis, caries, plaque and gum bleeding. Out of more than 700 oral microbial species, some opportunistic pathogens such as Staphylococcus aureus, Streptococcus spp. and Candida albicans are more prevalent. In this study, the antimicrobial activities of various toothpastes (dilutions ranging from 1:1-1:128) against above mentioned pathogens were assessed. The pathogens were isolated from clinical samples using various differential and selective media and identified through microscopic examination, cultural characteristics and biochemical tests using both conventional and API kit system (Biomerieux, France). Antimicrobial activities of selected dentifrice formulations against identified microbes were determined using agar well diffusion and Minimum Inhibitory Concentration assays. Statistical analysis of the data on different variables has been performed by Analysis of Variance and Mean ±SD using SPSS software. From the collected samples Staphylococcus aureus, Streptococcus mutans, Streptococcus salivarius, Streptococcus intermedius and Candida albicans were isolated and identified. All the selected toothpastes showed significant (p<0.01) antimicrobial activity against the bacterial and fungal isolates. Variable results (inhibitory zone diameters ranging from 35.10±8.00 to 2.40±5.37) were found when mean of different dilutions were compared. Conventional dentifrices exhibited more inhibition as compared to herbal products.

Molecular detection of blaVIM Metallo-ß-lactamase producing clinically isolated Pseudomonas aeruginosa from tertiary care hospital, Faisalabad.

Metallo-ß-lactamases (MBLs) producing Pseudomonas aeruginosa are major threat for public health. They produce resistance against various antibiotics and remain low or no therapeutic options. A total of 200 clinical isolates of P. aeruginosa were collected from tertiary care hospital, Faisalabad. Isolates were sub-cultured on basic and selective media and confirmed by API 20NE. Phenotypic detection of carbapenamase, MBLs, antibiogram and MIC were determined as per CLSI guidelines. Molecular detection of blaVIM was performed using specific primers by PCR. Among 200 P. aeruginosa, majority (n=82) were isolated from pus samples followed by 28 from tracheal aspirates and 27 from sputum. Out of 110 (55%) MDR P. aeruginosa, 12 (11%) were positive for MHT and MBLs and blaVIM was identified in MBL positive isolates. Antibiogram revealed that all the isolates were resistant to ß-lactam drugs including carbapenems followed by 95% to levofloxacin, 67% to doxycycline and more effective drugs were tigecycline and colistin. MIC value for imipenem drug was 16µg/mL and 8µg/mL against 6 and 5 isolates respectively while MIC value for meropenem against 6 and 3 isolates were 8µg/mL and 16µg/mL respectively. Our study concluded the high prevalence of blaVIM producing P. aeruginosa in our clinical settings.

Biosurfactants production potential of native strains of Bacillus cereus and their antimicrobial, cytotoxic and antioxidant activities.

Present study was designed to evaluate the biosurfactant production potential by native strains of Bacillus cereus as well as determine their antimicrobial and antioxidant activities. The strains isolated from garden soil were characterized as B. cereus MMIC 1, MMIC 2 and MMIC 3. Biosurfactants were extracted as grey white precipitates. Optimum conditions for biosurfactant production were 37°C, the 7th day of incubation, 0.5% NaCl, pH 7.0. Moreover, corn steep liquor was the best carbon source. Biuret test, Thin Layer Chromatography (TLC), agar double diffusion and Fourier Transform Infrared Spectroscopy (FTIR) characterized the biosurfactants as cationic lipopeptides. Biosurfactants exhibited significant antibacterial and antifungal activity against S. aureus, E. coli, P. aeruginosa, K. pneumoniae, A. niger and C. albicans at 30 mg/ml. Moreover, they also possessed antiviral activity against NDV at 10 mg/ml. Cytotoxicity assay in BHK-21 cell lines revealed 63% cell survival at 10 mg/ml of biosurfactants and thus considered as safe. They also showed very good antioxidant activity by ferric-reducing activity and DPPH scavenging activity at 2 mg/ml. Consequently, the study offers an insight for the exploration of new bioactive molecules from the soil. It was concluded that lipopeptide biosurfactants produced from native strains of B. cereus may be recommended as safe antimicrobial, emulsifier and antioxidant agent.

Molecular basis of quinolone resistance in clinical isolates of Klebsiella pneumoniae from Pakistan.

Antibiotic resistant Klebsiella pneumoniae, is associated with various nosocomial infections that are difficult to treat. This study is designed to find out the patterns of resistance against commonly used antibiotics in K. pneumoniae clinical isolates with special attention to fluoroquinolones. A total number of 200 K. pneumoniae clinical isolates collected from various tertiary care hospitals of Punjab, Pakistan for a span of 1 year were investigated. Isolates were identified biochemically and genetically using VITEK® system and species-specific PCR, respectively. Antibiogram of isolates was studied by using disc diffusion and broth micro-dilution assays. Highest infection of K. pneumoniae detected in urinary tract (43%) followed by respiratory tract (25.5%). Most of the isolates displayed strong resistance against ampicillin, cefotetan, tazobactam, cefuroxime, cefixime, ceftriaxone, ampicillin-sulbactam imipenem, meropenem, ciprofloxacin and moxifloxacin, while sensetive to cefotaxime. Chromosoaml mutation was deteted in gyrA gene, gyrA harbors a strong mutation which provides resistance against ciprofloxacin by substituting Ser83→Ile. However, no mutation was detected in gyrB gene. Moreover, qnrB1 plasmid born resistant gene was only detected among qnrA, qnrB and qnrS. The story depicts an alarming situation of antibiotic resistance among K. pneumoniae associated with various nosocomial infections.

Effect of Lactobacillus casei on serum interleukins following enteropathogenic E. coli infection in experimental rabbits.

In the present study we investigated the pro-inflammatory and anti-inflammatory effect of Lactobacillus casei following infection with multi-drug resistant enteropathogenic Escherichia coli infection in experimental rabbits. For this purpose, 40 adult rabbits were divided into different groups and were infected with multi-drug resistant E. coli AZ1 strain except the control groups. The rabbits were orally administered with L. casei SABA6 strain in two different ways i.e. pre-treatment and post-treatment and both were continued for 7 days. The rabbits were sacrificed sequentially at 0, 4, 7 and 10 days post infection (dpi). Serum and intestinal tissue samples were collected from each rabbit. Intestinal tissue samples were subjected to histopathological examination that showed microscopic lesions at 4 and 7 dpi among infected group. The serum samples were processed for determination of Interleukin-6 (IL-6, pro-inflammatory) and Interleukin-10 (IL-10, anti-inflammatory) using ELISA. It was found that oral administration of L. casei SABA6 reduces the eruption of intestinal epithelial cells and reduces the incidence of diarrhea. Further, L. casei SABA6 also resulted in immuno modulation by significant increase in concentration of IL-6 and IL-10 particularly at 4 and 7 dpi and protects against E. coli AZ1 infection. Altogether, it was concluded that increased IL-6 and IL-10 levels were responsible for protection against EPEC infections. The sequential sacrifice of experimental animals could be adopted for future studies to find out pathogenesis and virulence mechanism of EPEC infections along with protective efficacy of different probiotics.

Efficacy of Azadirachta indica organic extracts against clinical methicillin resistant Staphylococcus aureus isolates.

In current study we investigated the efficacy of organic extracts of Azadirachta indica leaves against Methicillin Resistant Staphylococcus aureus (MRSA) clinical isolates. For this purpose fresh leaves were used to prepare ethanol, methanol and chloroform extract. Secondly, a cross sectional study was conducted to isolate MRSA in clinical samples from patients having surgical/ non-surgical wounds from Allied Hospital and District Head Quarter Hospital, Faisalabad. The S. aureus isolates were initially identified by biochemical characterization, followed by identification of MRSA using cefoxitin disc diffusion test that was finally confirmed by genomic amplification of mecA gene, responsible for resistance. All MRSA isolates were tested to find vancomycin resistant S. aureus (VRSA) using E-strips (M.I.C. EvaluatorTM, Oxide, UK). The data showed an overall 37% prevalence of S. aureus including 56.75% clinical MRSA isolates while none of the isolated S. aureus showed resistance to vancomycin. The antimicrobial activity was measured as mean zone of inhibition for each extract against all MRSA isolates and it was found as 15.38±2.26, 16.09±3.09 and 17.42±2.48 for methanol, ethanol and chloroform extracts respectively. Chloroform extract showed significantly high antimicrobial activity against MRSA isolates. Altogether, the current study exposed the high prevalence of MRSA isolates from tertiary care hospitals. However, all MRSA isolates were found susceptible to organic extracts of A. indica leaves.

Comparative evaluation of antibacterial activity of foot muscle extracts from genus Physa and genus Ceciloides (Mollusca: Gastropoda).

The aim of this study is to explore the presence of antimicrobial bioactive agents in the foot muscle extracts of snails belonging to genus Physa and Ceciloides. Antibacterial activity of foot extracts belonging to species named as P. fontinalis, P. gyrina, P. acuta, C. acicula, C. eulima, C. petitiana, was checked and compared against three bacterial strains i.e. E.coli, P. auroginosa, S. aureus by using disc diffusion method. The results were highly significant with maximum zone of inhibition of 20.10 mm in the P. fontinalis acetone extract and the least was 12.97 mm of C. eulima diethyl ether extract. The microdilution method was employed to observe MIC to evaluate antimicrobial resistance pattern of snails foot muscle extract against three mentioned strains. MIC of foot extracts was ranging from 0.03µ/ml-5 µg/ml for six species. TLC was carried out for profiling of extracts with positive results. Foot extracts from species of both genera eluted in different fractions of compounds with a good resolution in 100% n-hexane and ethyl acetate each. The plates developed in solvent system showed purple and yellow spots indicating the presence proteins and organic compounds showing it a promising canditadate for the therapeutic purposes.

Exploring the antimicrobial efficacy of Manuka honey against multidrug-resistant and extensively drug-resistant Salmonella Typhi causing septicemia in Pakistan.

Aim: To determine the efficacy of manuka honey against multidrug-resistant (MDR) and extensively drug-resistant (XDR) clinical strains of Salmonella Typhi. Materials & methods: Clinical isolates were processed using the Bactec blood culture system, identification and antibiogram by Vitek 2 and antibiotic resistance genes through polymerase chain reaction (PCR). Microbroth dilution assays evaluated the antibacterial activity of manuka honey. Results: MDR and XDR-S. Typhi was susceptible to azithromycin. These strains carried the H58, gyrA, gyrB, blaCTX-M-15 , and blaTEM-1 genes. At 100% honey, the zone of inhibition for MDR (15-23 mm) and XDR (15-24 mm) strains. 18/50 MDR and 14/50 XDR strains inhibited at 3.125 v/v% killed at 6.25 v/v% concentration respectively. Conclusion: Manuka honey could be an alternative option for treating S. Typhi infections.

Typhoid fever is a life-threatening bacterial infection caused by the Salmonella Typhi. These bacteria are transmitted through contaminated water and food and cause fever, abdominal pain, headache, vomiting, and diarrhea mainly in children under 5. There are around 9 million people get infected with S. Typhi, with an increased death of 1,10,000 annually. Bees that collect nectar from the blossoms of the Manuka tree in Australia and New Zealand produce a type of honey known as manuka honey. This honey is famous for its antibacterial activity, and potential health benefits. Therefore, we aimed to determine its antibacterial activity against S. Typhi. Our finding shows that the commonly available antibiotics did not kill S. Typhi because their DNA was drug-resistant. After applying the manuka honey, these bacteria were killed and given a clear zone ranging from 15­24mm on the agar plate. Further analysis revealed that at low concentrations of manuka honey, 3.1% and 6.25%, most of the S. Typhi stopped growing and killed, respectively. This study suggested that manuka honey, which is affordable and readily available, could be used as a treatment option to treat infections produced by these harmful bacteria after further analysis.

A Health Threat from Farm to Fork: Shiga Toxin-Producing Escherichia coli Co-Harboring blaNDM-1 and mcr-1 in Various Sources of the Food Supply Chain.

The dissemination of resistant pathogens through food supply chains poses a significant public health risk, spanning from farm to fork. This study analyzed the distribution of Shiga toxin-producing Escherichia coli (STEC) across various sources within the animal-based food supply chain. A total of 500 samples were collected from livestock, poultry, the environment, fisheries, and dairy. Standard microbiological procedures were employed to isolate and identify E. coli isolates, which were further confirmed using MALDI-TOF and virulence-associated genes (VAGs) such as stx1, stx2, ompT, hylF, iutA, fimH, and iss. The phenotypic resistance patterns of the isolates were determined using the disc diffusion method, followed by molecular identification of antibiotic resistance genes (ARGs) through PCR. STEC were subjected to PCR-based O typing using specific primers for different O types. Overall, 154 (30.5%) samples were confirmed as E. coli, of which 77 (50%) were multidrug-resistant (MDR) E. coli. Among these, 52 (67.53%) isolates exhibited an array of VAGs, and 21 (40.38%) were confirmed as STEC based on the presence of stx1 and stx2. Additionally, 12 out of 52 (23.07%) isolates were identified as non-O157 STEC co-harbouring mcr-1 and blaNDM-1. O26 STEC was found to be the most prevalent among the non-O157 types. The results suggest that the detection of STEC in food supply chains may lead to serious health consequences, particularly in developing countries with limited healthcare resources.

Co-Occurrence of mcr-1 and Carbapenem Resistance in Avian Pathogenic E. coli Serogroup O78 ST95 from Colibacillosis-Infected Broiler Chickens.

Avian pathogenic Escherichia coli (APEC) is responsible for significant economic losses in the poultry industry. This study aimed to molecularly detect carbapenem-resistant co-harboring mcr-1 avian pathogenic E. coli in broiler chickens infected with colibacillosis. A total of 750 samples were collected from colibacillosis-infected broilers, and conventional microbiological techniques were used to isolate and identify APEC. MALDI-TOF and virulence-associated genes (VAGs) were used for further identification. Phenotypic carbapenem resistance profiling was followed by molecular detection of carbapenem resistance genes (CRGs) and other resistance genes through PCR using specific primers. Isolates were also subjected to PCR for O typing, followed by allele-specific PCR to detect sequence type (ST) 95. Results showed that 154 (37%) isolates were confirmed as APEC, with 13 (8.4%) isolates found to be carbapenem-resistant (CR)-APEC. Among CR-APEC isolates, 5 (38%) were observed to co-harbor mcr-1. All CR-APEC showed the presence of five markers (ompT, hylF, iutA, iroN, and iss) APEC VAGs, and 89% of CR-APEC isolates displayed O78 type. Furthermore, 7 (54%) CR-APEC isolates were observed with ST95, all displaying O78 type. These results suggest that the improper use of antibiotics in poultry production systems is contributing to the emergence of pathogens such as CR-APEC co-harboring the mcr-1 gene.

Insights into the Intersection of Biocide Resistance, Efflux Pumps, and Sequence Types in Carbapenem-Resistant Acinetobacter baumannii: A Multicenter Study.

Acinetobacter baumannii, a pathogenic bacterium acquired in hospitals, causes diverse infections in humans. Previous studies have reported resistance among A. baumannii strains, potentially selecting multi-drug-resistant variants. In Pakistan, research has primarily focused on carbapenem-resistant A. baumannii (CRAB) strains, overlooking the investigation of efflux pumps (EPs) and biocide resistance. This study aims to assess A. baumannii strains from five hospitals in Pakistan, focusing on antibiotic and biocide susceptibility, the impact of EP inhibitors on antimicrobial susceptibility, and the distribution of ARGs and STs. A total of 130 non-repeated Acinetobacter baumannii isolates were collected from five tertiary care hospitals in Pakistan and identified using API 20NE and multiplex PCR. Antimicrobial susceptibility testing utilized disc diffusion and broth microdilution assays, while biocide susceptibility was assessed with various agents. The impact of an efflux pump inhibitor (NMP) on antibiotic susceptibility was evaluated. PCR screening for ARGs and EPGs was followed by DNA sequencing validation. MLST was performed using the Pasteur scheme. Most isolates demonstrated resistance to tested antibiotics, with varying levels of susceptibility to biocides. All isolates exhibited the intrinsic class D ß-lactamase blaOXA-51, while acquired blaOXA-23 was present in all CRAB isolates. Among EPs, adeJ, abeD, amvA, and aceI were prevalent in almost all isolates, with adeB found in 93% of isolates and adeG, adeT1, adeT2, and qacEΔ1 displaying lower prevalence ranging from 65% to 79%. The most common STs were ST589 and ST2, accounting for 28.46% and 25.38% of isolates, respectively, followed by ST642 at 12.6%. These findings indicate that A. baumannii strains in Pakistan are resistant to antibiotics (excluding colistin and tigecycline) and may be developing biocide resistance, which could contribute to the selection and dissemination of multi-drug-resistant strains.

Penicillin production by wild isolates of Penicillium chrysogenum in Pakistan.

The present study was aimed at exploring the native wild isolates of Penicillium chrysogenum series in terms of their penicillin production potential. Apart from the standard medium, the efforts were made to utilize suitable agro-industrial wastes for the maximum yield of penicillin. Two series of P. chrysogenum were isolated from local sources and named as P. chrysogenum series UAF R1 and P. chrysogenum series UAF R2. The native series were found to possess better penicillin production potential than the already reported series of P. chrysogenum. However, P. chrysogenum series UAF R1 was found to be the best candidate for high yield of penicillin starting at 100 hour as compared to P. chrysogenum series UAF R2 which produced the highest yield of penicillin at 150 hours for a shorter period of time. Addition of Corn Steep Liquor (CSL) to the fermentation medium resulted in the production of 1.20g/L penicillin by P. chrysogenum series UAF R1 and P. chrysogenum series UAF R2. The fermentation medium in which Sugar Cane Bagasse (SCB) was replaced with CSL resulted in the highest yield of penicillin (1.92g/L) by both native series of P. chrysogenum. The penicillin production was increased by 62.5% in medium with SCB as compared to that with CSL. The penicillin yield of medium containing lactose and phenyl acetate was higher than that of control medium. Overall results revealed that P. chrysogenum series UAF R1 and P. chrysogenum series UAF R2 may be recommended for better yield of natural penicillin and this efficiency may be further enhanced by utilizing SCB as substrate in the growth medium.

Multilocus Sequence Typing of Carbapenem-Resistant Acinetobacter baumannii Isolates Harboring blaOXA-23 and blaIMP in Cattle from Punjab, Pakistan.

Acinetobacter baumannii is a notorious bacterial pathogen that can cause an array of nosocomial infections in clinical settings. However, the data from the veterinary settings is limited and especially in Pakistan, no such study is conducted so far. To investigate the prevalence, antimicrobial resistance, and distribution of specific sequence types of A. baumannii in cattle, a total of 1,960 samples were collected from cattle over 18 months from Punjab, Pakistan. The isolates obtained were identified using the API20NE system and confirmed through PCR. The isolated A. baumannii isolates were further screened for antimicrobial susceptibility and the presence of resistance genes. Multilocus sequence typing was carried out to characterize the carbapenem-resistant A. baumannii (CRAB) isolates. Results revealed an overall prevalence of A. baumannii at 3.31% (65/1,960) with a higher prevalence of 7.38% (54/731) in dairy cattle compared to beef cattle at 4.41% (11/249). Among 65 A. baumannii isolates, 27.7% (18/65) were CRAB. All CRAB isolates harbor class D ß-lactamases genes blaOXA-23 and blaOXA-51, whereas 94.4% (17/18) CRAB isolates carried class B ß-lactamases gene blaIMP, and only one isolate had blaNDM-1 gene. The commonly found sequence types for CRAB isolates were ST2 and ST642 corresponding to 10 and 05 isolates, respectively. The presence of CRAB in cattle indicates an alarming situation that necessitates an urgent and efficient surveillance system to limit the transmission of CRAB among the cattle population and its possible transmission to humans and the environment.