Effectiveness of cephalosporins in hydrolysis and inhibition of Staphylococcus aureus and Escherichia coli biofilms.

IMPORTANCE: Staphylococcus aureus and Escherichia coli contribute to global health challenges by forming biofilms, a key virulence element implicated in the pathogenesis of several infections. OBJECTIVE: The study examined the efficacy of various generations of cephalosporins against biofilms developed by pathogenic S. aureus and E. coli. METHODS: The development of biofilms by both bacteria was assessed using petri-plate and microplate methods. Biofilm hydrolysis and inhibition were tested using first to fourth generations of cephalosporins, and the effects were analyzed by crystal violet staining and phase contrast microscopy. RESULTS: Both bacterial strains exhibited well-developed biofilms in petri-plate and microplate assays. Cefradine (first generation) showed 76.78% hydrolysis of S. aureus biofilm, while significant hydrolysis (59.86%) of E. coli biofilm was observed by cefipime (fourth generation). Similarly, cefuroxime, cefadroxil, cefepime, and cefradine caused 78.8%, 71.63%, 70.63%, and 70.51% inhibition of the S. aureus biofilms, respectively. In the case of E. coli, maximum biofilm inhibition (66.47%) was again shown by cefepime. All generations of cephalosporins were more effective against S. aureus than E. coli, which was confirmed by phase contrast microscopy. CONCLUSIONS AND RELEVANCE: Cephalosporins exhibit dual capabilities of hydrolyzing and inhibiting S. aureus and E. coli biofilms. First-generation cephalosporins exhibited the highest inhibitory activity against S. aureus, while the third and fourth generations significantly inhibited E. coli biofilms. This study highlights the importance of tailored antibiotic strategies based on the biofilm characteristics of specific bacterial strains.

Molecular identification of different toxinogenic strains of Clostridium perfringens and histo-pathological observations of camels died of per-acute entero-toxaemia.

Enterotoxaemia is a severe disease caused by Clostridium perfringens and render high mortality and huge economic losses in livestock. However, scanty information and only few cases are reported about the presence and patho-physiology of enterotoxaemia in camels. The bacterium induces per-acute death in animals due to rapid production of different lethal toxins. The necropsy of camels (per-acute = 15, acute = 3) was conducted at 18 outbreaks of enterotoxaemia in camels in the desert area of Bahawalpur region. At necropsy, the serosal surfaces of visceral organs in the abdominal, peritoneal and thoracic cavities were found to have petechiation with severe congestion. Moreover, both the cut-sections of different visceral organs and the histo-pathological analysis revealed the pathological lesions in heart, lungs, kidneys, spleen, small and large intestines. Grossly, the kidneys were severely congested, hyperemic, swollen and softer in consistency. Under the microscope, different sections of kidneys indicated that the convulated and straight tubules were studded with erythrocytes. In the intestines, there were stunting fusion of crypts and villi. Similarly, various histo-pathological ailments were also observed in the heart, lungs and spleen. At blood agar, the collected samples showed beta hemolytic colonies of C. perfringens that appeared as medium sized rods microscopically and stained positively on Gram staining. Multiplex PCR revealed C. perfringens type A (α and ß2 genes) and D (epsilon gene) and the deaths were found to be significantly higher due to C. perfringens type D compared to those by C. perfringens type A. Hence, it has been concluded that enterotoxaemia in camel affects multiple organs and becomes fatal, if occurred due to C. perfringens type D.

Occurrence of Virulence Genes among Methicillin-Resistant Staphylococcus aureus Isolated from Subclinical Bovine Mastitis.

The occurrence of Staphylococcus aureus-induced subclinical mastitis holds significant implications for public health. This specific microorganism possesses a wide array of pathogenic factors that enable it to adhere to, colonize, invade, and infect the host. The objective of the current study was to assess the prevalence of S. aureus, determine antimicrobial resistance patterns, and identify virulence genes of methicillin-resistant S. aureus (MRSA) strains responsible for subclinical mastitis in bovines. A total of 249 milk samples were collected from various farms in the district of Faisalabad. The presence of subclinical mastitis was assessed by using the California mastitis test. Positive milk samples (n = 100) were then subjected to standard microbiological techniques for isolation and identification of S. aureus. Antibiogram analysis was conducted by using the disc diffusion method to assess antimicrobial resistance. For the molecular detection of S. aureus and its virulence genes, the polymerase chain reaction (PCR) was performed with species-specific primers. The overall prevalence of S. aureus was found to be 40% (40/100), which was confirmed through molecular detection of the nuc gene in 40/40 (100%) of samples using PCR. Antimicrobial susceptibility tests indicated the highest susceptibility to vancomycin, sulfamethoxazole/trimethoprim, erythromycin, gentamicin, ciprofloxacin, and chloramphenicol, while the highest resistance rate was observed against tetracycline. Additionally, 30% of samples (12/40) tested positive for methicillin resistance. PCR analysis revealed that 100% of MRSA-tested isolates harbored the mecA and clfA genes. Furthermore, the MRSA isolates showed the presence of pvl, hla, hlb, sec, icaA, icaD, icaB, and icaC genes at rates of 92, 75, 67, 42, 42, 75, 8, and 25%, respectively. These findings underscore the need for stricter aseptic control in dairy farms to prevent disease transmission between animals and ensure the production of safe and uncontaminated food for human consumption.

Synthesis and Characterization of Silica, Silver-Silica, and Zinc Oxide-Silica Nanoparticles for Evaluation of Blood Biochemistry, Oxidative Stress, and Hepatotoxicity in Albino Rats.

Evaluation of nanoparticles (NPs) for biomedical applications has received a lot of attention for detailed study on pharmacokinetics prior to clinical application. In this study, pure C-SiO2 (crystalline silica) NPs and SiO2 nanocomposites with silver (Ag) and zinc oxide (ZnO) were prepared by utilizing different synthesis routes such as sol-gel and co-precipitation techniques. The prepared NPs showed highly crystalline nature as confirmed by X-ray diffraction analysis where average crystallite sizes of 35, 16, and 57 nm for C-SiO2, Ag-SiO2, and ZnO-SiO2 NPs, respectively, were calculated. Fourier transform infrared analysis confirmed the presence of functional groups related to the chemicals and procedures used for sample preparation. Due to agglomeration of the prepared NPs, the scanning electron microscope images showed large particle sizes when compared to their crystalline sizes. The optical properties of the prepared NPs such as absorption were obtained with UV-Vis spectroscopy. For in vivo biological evaluation, albino rats, both male and female, kept in different groups were exposed to NPs with 500 µg/kg dose. Hematological, serum biochemistry, histo-architecture, oxidative stress biomarkers, and antioxidant parameters in liver tissues along with various biomarkers for the evaluation of erythrocytes were estimated. The results on hemato-biochemistry, histopathological ailments, and oxidative stress parameters exhibited 95% alteration in the liver and erythrocytes of C-SiO2 NPs-treated rats while 75 and 60% alteration in the liver tissues of rats due to exposure to Ag-SiO2 and ZnO-SiO2 NPs, respectively, when compared with the albino rats of the control (untreated) group. Therefore, the current study showed that the prepared NPs had adverse effects on the liver and erythrocytes causing hepatotoxicity in the albino rats in respective order C-SiO2 > Ag SiO2 > ZnO-SiO2. As the C-SiO2 NPs appeared to be the most toxic, it has been concluded that coating SiO2 on Ag and ZnO reduced their toxicological impact on albino rats. Consequently, it is suggested that Ag-SiO2 and ZnO-SiO2 NPs are more biocompatible than C-SiO2 NPs.

Distribution of mcr-1 Harboring Hypervirulent Klebsiella pneumoniae in Clinical Specimens and Lytic Activity of Bacteriophage KpnM Against Isolates.

Background: The World Health Organization (WHO) has declared the multi-drug resistant (MDR) Klebsiella pneumoniae as one of the critical bacterial pathogens. The dearth of new antibiotics and inadequate therapeutic options necessitate finding alternative options. Bacteriophages are known as enemies of bacteria and are well-recognized to fight MDR pathogens. Methods: A total of 150 samples were collected from different clinical specimens through a convenient sampling technique. Isolation, identification, and antibiotic susceptibility testing (AST) of K. pneumoniae were done by standard and validated microbiological procedures. Molecular identification of virulence factors and antibiotic resistance genes (ARGs) was carried out through polymerase chain reaction (PCR) by using specific primers. For bacteriophage isolation, hospital sewage samples were processed for phage enrichment, purification, and further characterization ie, transmission electron microscopy (TEM) and stability testing, etc. followed by evaluation of the lytic potential of the phage. Results: Overall, a total of 41% of isolates of K. pneumoniae were observed as hypervirulent K. pneumoniae (hvKp). Among hvKp, a total of 12 (42%) were detected as MDR hvKp. A total of 37% of all MDR isolates were found resistant to colistin, and 66% of the colistin resistance isolates were recorded as mcr-1 positive. Isolated phage KpnM had shown lytic activity against 53 (79%) K. pneumoniae isolates. Remarkably, all 8 mcr-1 harboring MDR hvKp and non-hvKp isolates were susceptible to KpnM phage. Conclusion: Significant distribution of mcr-1 harboring hypervirulent Klebsiella pneumoniae was observed in clinical specimens, which is worrisome for the health system of the country. Characterized phage KpnM exhibited encouraging results and showed the lytic activity against the mcr-1 harboring hvKp isolates, which may be used as a prospective alternative control strategy to fight this ominous bacterium.

Genetic Diversity of Escherichia coli Coharboring mcr-1 and Extended Spectrum Beta-Lactamases from Poultry.

Background: The emergence of resistance to beta-lactam agents in poultry results in multidrug-resistant (MDR) phenotypes in Escherichia coli isolates from poultry birds. The appearance of mobile colistin resistance (mcr) genes in the poultry sector has further worsened the situation. Therefore, the current study is aimed at investigating the molecular epidemiology of mcr harboring colistin-resistant E. coli among poultry. Methods: The isolation and identification of colistin-resistant E. coli (CR-Ec) were done from the broiler's fecal samples through culturing using selective media supplemented with colistin sulfate (4 µg/ml). The antibiogram studies of the isolates were performed using the disc diffusion method and broth microdilution method as per CLSI guidelines. The screening for the genes conferring resistance to colistin as well as beta-lactam agents was performed by PCR. The genetic diversity of mcr-positive strains was assessed by multilocus sequencing typing (MLST). Results: Out of 500 fecal samples, 7% (35/500) were found positive for the presence of colistin-resistant E. coli (CR-Ec). Among the CR-Ec isolates, 74.28% (26/35) were detected as ESBL producers carrying the blaCTX-M-1 gene in 15/35 (42.85%) isolates and blaCTX-M-15 and blaTEM genes in 21/35 (60%) and 35/35 (100%) isolates, respectively. E. coli isolates were found positive for the presence of mcr-1, although none of the isolates exhibited the mcr-2 or mcr-3 genes. The MLST of CR-Ec has shown the ST1035 as the most prevalent genotype, while 82.85% (29/35) of CR-Ec strains belonged to clonal complex (CC) 131 comprising ST1035, ST131, ST1215, ST1650, and ST2279. Conclusions: The findings suggest a continuous monitoring system in veterinary and clinical settings to avoid unnecessary antibiotics. Further studies are needed at the national level to help control the increasing resistance among Enterobacterales in poultry settings.

Distribution and genetic diversity of multi-drug-resistant Klebsiella pneumoniae at the human-animal-environment interface in Pakistan.

Klebsiella pneumoniae is ubiquitous and known to be a notorious pathogen of humans, animals, and plant-based foods. K. pneumoniae is a recognized trafficker of antibiotic resistance genes (ARGs) between and from different ecological niches. A total of 775 samples (n = 775) were collected from September 2017 to August 2019 from humans, animals, and environmental sources by applying the random convenient sampling technique. A total of 120 (15.7%) samples were confirmed as K. pneumoniae. The distribution of K. pneumoniae among humans, the environment, and animals was 17.1, 12.38, and 10%, respectively. Isolates have shown significant resistance against all the subjected antibiotics agents except colistin. ARGs profiling revealed that the highest percentage prevalence (67.5%) of bla CTX-M was estimated in the isolates, and various carbapenem resistance genes that were found in the study were bla NDM-1 (43.3%), bla OXA-48 (38%), and (1.67%) bla KPC-2. Overall, 21 distinct sequence types (ST) and 13 clonal complexes (CCs) were found through the multi-locus sequence typing (MLST) analysis. Taking together, the distribution of multi-drug resistance (MDR) K. pneumoniae clones in the community and associated environment is alarming for the health care system of the country. Health policymakers should consider the role of all the integral parts of humans, animals, and the associated environment intently to cope with this serious public and animal health concern.

Histopathological Investigations and Molecular Confirmation Reveal Mycobacterium bovis in One-Horned Rhinoceros (Rhinoceros unicorns).

Mycobacterium bovis causes tuberculosis in dairy and wild animals. Presence of tuberculosis in animals poses a threat not only to their herd mates but also for public. No reports are available about the clinical, pathological, and molecular investigation of naturally occurring tuberculosis (TB) due to M. bovis in one-horned rhinoceros. One-horned female rhinoceros (Rhinoceros unicorns) at the age of 41 years died in a public park in Pakistan. Postmortem and other investigations were carried out to know the cause of death. The present study describes necropsy, histopathology, and molecular-based confirmation of TB in a captive female rhinoceros that died of this infection. Clinically, the rhinoceros showed nonspecific clinical signs including anorexia, lethargy, dyspnoea, coughing, and sudden death. At necropsy, the trachea exhibited mild congestion and contained catarrhal exudate at the bronchial bifurcation. Macroscopic examination revealed characteristic tubercles on all parenchymatous organs. The lungs showed consolidation, grey hepatization, and contained granulomatous lesions packed with cheesy exudate. Histopathological examination showed severe pneumonic changes in the form of granulomatous inflammation consisting of lymphocytes, multinucleated giant cells, caseous materials, and mineralized foci surrounded by a fibrous capsule. PCR amplicon of 500 bp confirmed the presence of M. bovis in multiple hepatic and pulmonary tissue samples, as well as in uterine exudates. It was concluded that the presence of tuberculosis in rhinoceros may pose potential transmission risk to other animals and the application of practical tools to determine TB status in the rhinoceros is crucial.

Pathological, Histological, and Molecular Based Investigations Confirm Novel Mycobacterium bovis Infection in Boselaphus tragocamelus.

Mycobacterium bovis (M. bovis) being the main cause of animal tuberculosis is a complex infectious agent and can be a cause of zoonotic tuberculosis zoonosis in public health. To date, the uncommon infection in public health due to M. bovis still is a great challenge to both veterinary and medical professions and requires a careful diagnosis and confirmation of the bacterium. Therefore, this study for the first time reports the clinical, gross, histopathological, and molecular based confirmation of M. bovis infection in wildlife animals (nilgai). Prior to death, the morbid animal showed severe pneumonic ailments like moist cough, thick nasal exudates, and dyspnoea. At necropsy, enlargement of mandibular cervical and mesenteric lymph nodes was observed. Different macroscopic lesions such as congestion and hyperaemia, creamy white and catarrhal exudates in trachea, consolidation, grey and red hepatisation of lungs, and micro- and macrogranulomatous tubercles containing caseous materials in lungs were observed. The heart of morbid animal showed congestions, myocarditis, and a copious amount of straw-colored fluid in the pericardial sac. At the microscopic level, lungs indicated granulomatous inflammatory response, presence of multinucleated giant cells, fibrosis, and punctuation of alveoli with chronic inflammatory cells. Histopathological examination of various sections of the heart of the infected animal showed chronic inflammatory response consisting of chronic inflammatory cells like monocyte, lymphocytes, and fibroblasts along with noncalcified eosinophilic materials. At the molecular level, M. bovis infection was confirmed in various tissues like the heart, lungs, cervical, and mesenteric lymph nodes in morbid animals. In conclusion, based on our results, it can be suggested that more molecular based epidemiological studies are crucial to know the exact cause of pulmonary and cervical tuberculosis in wild animals.

Clostridium perfringens Types A and D Involved in Peracute Deaths in Goats Kept in Cholistan Ecosystem During Winter Season.

Enterotoxemia is a severe and peracute disease caused by Clostridium perfringens (C. perfringens) rendering high mortality leading to huge economic losses, especially in small ruminants. The bacterium induces peracute death in animals based on the rapid production of different lethal toxins. Mortality occurred three private herds of two breeds, i.e., Makhi Cheeni and Beetal, and one non-descriptive (Teddy) herds reared in the desert area of Bahawalpur, Pakistan. At necropsy, tissue samples for histopathology and intestinal contents for bacterial isolation and culture were collected. Following the standard procedure, tissue slides were prepared. Multiplex PCR was used to identify toxinotypes using specific primers. Morbidity, mortality, and case fatality in Makhi Cheeni, Beetal, and Teddy goats caused by enterotoxemia were 87.58, 75.81, and 76.11%, respectively. Based on toxinotypes in the present outbreaks, C. perfringens type A (cpα = 20.7%; cpα + cpß2 = 11.2%) and C. perfringens type D (cpα + cpß2 + etx = 47.7%; cpα + etx = 20.7%) were detected. Deaths due to C. perfringens type D (68.10%) were significantly higher (p < 0.001) compared with deaths by C. perfringens type A (34.90%). Petechiation of serosal surfaces, hemorrhage of intestines, lungs, and liver were seen. Kidneys were soft, and under the microscope, tubules were studded with erythrocytes. There was stunting and fusion in the intestinal villi. From this study, we concluded that endotoxemia can occur in any season; thus, a proper vaccination schedule must be followed for the protection of small ruminants' health.

Antibacterial activity of silver nanoparticles against carbapenem-resistant Acinetobacter baumannii clinical isolates.

Carbapenem-resistant Acinetobacter baumannii (CRAB) produce resistance to various classes of antibiotics and left limited options for treatment. This study was designed to determine antibacterial activity of AgNPs against CRAB. Total 100 A. baumannii were collected from a tertiary care hospital, Lahore. Isolates were subcultured on blood and MacConkey agar. Preliminary identification was carried out by morphological and biochemical tests. Antibiogram was done by Kirby-Bauer disc diffusion method. Antibacterial activity of AgNPs was performed by agar well diffusion method, while minimum inhibitory concentration and minimum bactericidal concentration were determined by micro broth dilution assay. Of 100 A. baumannii, 24 were confirmed as carbapenem-resistant. These isolates were mainly recovered from tracheal secretion (8; 33%), CSF (5; 20.8%), and urine (4; 16.8%). Antibacterial activity of AgNPs revealed a maximum zone of inhibition, 22mm at 50mg/mL and 18mm at 40mg/mL by agar well diffusion method. MIC of AgNPs determined that 14 CRAB were inhibited at 12.5mg/mL and 7 at 25mg/mL. However, MBC revealed that 13 CRAB were killed at 25mg/mL and 7 at 50mg/mL. This study concluded that most of the CRAB were inhibited and killed at 12.5mg/mL and 25mg/mL, respectively. AgNPs can be used as an alternative therapeutic agent followed by their pharmacokinetics and pharmacognosy.

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.

Bacteriophage Proteome: Insights and Potentials of an Alternate to Antibiotics.

INTRODUCTION: The mounting incidence of multidrug-resistant bacterial strains and the dearth of novel antibiotics demand alternate therapies to manage the infections caused by resistant superbugs. Bacteriophages and phage=derived proteins are considered as potential alternates to treat such infections, and have several applications in health care systems. The aim of this review is to explore the hidden potential of bacteriophage proteins which may be a practical alternative approach to manage the threat of antibiotic resistance. RESULTS: Clinical trials are in progress for the use of phage therapy as a tool for routine medical use; however, the existing regulations may hamper their development of routine antimicrobial agents. The advancement of molecular techniques and the advent of sequencing have opened new potentials for the design of engineered bacteriophages as well as recombinant bacteriophage proteins. The phage enzymes and proteins encoded by the lysis cassette genes, especially endolysins, holins, and spanins, have shown plausible potentials as therapeutic candidates. CONCLUSION: This review offers an integrated viewpoint that aims to decipher the insights and abilities of bacteriophages and their derived proteins as potential alternatives to antibiotics.

Distribution and antimicrobial susceptibility profile of bacterial and fungal pathogens isolated from burn wounds in hospitalized patients.

OBJECTIVE: Current study was designed to isolate the pathogens from burn wounds and determine the antibiogram of these isolates. METHODS: A total of 85 samples were collected from burn patients with the history of different weeks of hospitalization in various public and private hospitals of Faisalabad during September 2017-July 2019 and shifted to Department of Microbiology, Government College University, Faisalabad for further processing. Isolation and identification of the pathogens was done through conventional microbiological procedures. Disc diffusion method was used for the determination of antibacterial and antifungal activity. RESULTS: A total of 40(91%) samples were found positive for the presence of bacterial or fungal pathogens. Commonly isolated pathogens were Staphylococcus aureus 15 (21.4%), Pseudomonas aeruginosa 15 (21.4%), Bacillus subtilis 11(15.7%), Escherichia coli 10(14.2%), Candida albicans 8(11.4%), Aspergillus flavus 6(8.5%) and Salmonella Typhi 5(7.1%). Highest resistance was found against S. aureus and P. aeruginosa. Cefotaxime was the least effective antibiotic, while Gentamicin and Amphotericin-B were the mosteffective antimicrobial drugs against bacterial and fungal pathogens, respectively. CONCLUSIONS: Taking together it was concluded that most isolated pathogen was S. aureus and P. aeruginosa followed by B. subtilis, E. coli, C. albicans, A. flavus and S. typhi from burn wound in hospitalized patients. Anti-biogram studies showed S. aureus and P. aeruginosa were the most resistant pathogens whereas S. typhi, C. albicans and A. flavus were susceptible to various commonly used antibiotics. Cefotaxime was the least effective antibiotic, while Gentamicin and Amphotericin-B were the most effective antimicrobial drugs against bacterial and fungal pathogens, respectively. It is suggested that alternate anti-microbial agents should be investigated to control the infections.

Characterization of Pathogenic Vibrio parahaemolyticus Isolated From Fish Aquaculture of the Southwest Coastal Area of Bangladesh.

Vibrio parahaemolyticus is a major foodborne pathogen responsible for significant economic losses in aquaculture and a threat to human health. Here, we explored the incidence, virulence potential, and diversity of V. parahaemolyticus isolates from aquaculture farms in Bangladesh. We examined a total of 216 water, sediment, Oreochromis niloticus (tilapia), Labeo rohita (rui), and Penaeus monodon (shrimp) samples from the aquaculture system where 60.2% (130/216) samples were positive for V. parahaemolyticus. Furthermore, we identified 323 V. parahaemolyticus strains from contaminated samples, 17 of which were found positive for trh, a virulence gene. Four isolates out of the 17 obtained were able to accumulate fluid in the rabbit ileal loop assay. The correlation between the contamination of V. parahaemolyticus and environmental factors was determined by Pearson correlation. The temperature and salinity were significantly correlated (positive) with the incidence of V. parahaemolyticus. Most of the pathogenic isolates (94.1%) were found resistant to ampicillin and amoxicillin. O8: KUT was the predominant serotype of the potentially pathogenic isolates. ERIC-PCR reveals genetic variation and relatedness among the pathogenic isolates. Therefore, this region-specific study establishes the incidence of potential infection with V. parahaemolyticus from the consumption of tilapia, rui, and shrimp raised in farms in Satkhira, Bangladesh, and the basis for developing strategies to reduce the risk for diseases and economic burden.

In vitro efficacy of Azadirachta indica leaf extract against methicillin resistant Staphylococci isolated from skin infection.

In this cross-sectional study, the isolation and identification of Methicillin Resistant Staphylococcus aureus (MRSA) and Methicillin Resistant S. epidermidis (MRSE) was described from skin infections (n=100). Initial isolation was done by conventional procedures followed by amplification/ sequence analysis of 16S rRNA. Methicillin resistance was determined using cefoxitin discs and resistant isolates were screened for mec-A gene followed by Minimum Inhibitory Concentrations (MIC) determination of vancomycin. In second phase, we investigated extract of Azadirachta indica leaves using Fourier Transformed Infrared Spectroscopy (FTIR-Spectroscopy) and investigated in vitro activity. Initially, total of 28 Staphylococci were identified. 16S rRNA gene sequence confirmed S. aureus (22), S. epidermidis (3) and S. saprophyticus (3) isolates. Cefoxitin discs showed (7/22) MRSA, (3/3) (MRSE) and none of the methicillin resistant S. saprophyticus. MRSA and MRSE isolates showed presence of mec-A gene. However, all isolates were sensitive to vancomycin MIC (0.5-2µg/mL) and sensitive to Linezolid. FTIR-Spectroscopy of A. indica indicated the presence of azadirachtin and nimbolinin. The mean zone of inhibition was measured 14.23±1.37 and 13.66±0.70 against MRSA and MRSE isolates, respectively. Altogether, MRSA and MRSE is significant public health concern. However, vancomycin and linezolid were found effective and extract of A. indica showed in vitro effects.

Association of biofilm formation with multi drug resistance in clinical isolates of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is considered as foremost cause of hospital acquired infections due to its innate and plasmid mediated resistance to multiple antibiotics making it a multi drug resistant (MDR) pathogen. Biofilm formation is a pathogenic mechanism harbored by this pathogen which further elevates its resistance to antibiotics and host defense system. The aim of the present study was to evaluate the biofilm forming potential and distribution of pslA gene in multi drug resistant Pseudomonas aeruginosa isolates obtained from different clinical samples. A total of 200 different clinical samples were collected after obtaining written consent from the patients. The samples were subjected to isolation and identification of P. aeruginosa by standard microbiological procedures. Confirmation of isolates was done by polymerase chain reaction targeting oprL gene. Kirby Bauer method was performed for detection of MDR isolates. Congo red agar (CRA) test and Microtiter plate assay (MPA) for observing the biofilm forming ability and amplification of pslA gene was also performed on MDR isolates. The results showed that from 200 samples 52 (26 %) were P. aeruginosa and among them 20 (38.46 %) were MDR isolates. The CRA showed 23 (44.23 %) while MPA detected 49 (94.23 %) isolates as biofilm producers while all the MDR isolates showed biofilm formation by MPA method. The pslA gene was detected in all biofilm forming isolates while 90 % in MDR P. aeruginosa. It was concluded that biofilm forming P. aeruginosa are more resistant to tested antibiotics and biofilm formation is strongly associated with presence of pslA gene.

Molecular identification of blaCTX-M and blaTEM genes among multi-drug resistant Enteropathogenic Escherichia coli isolated from children.

In this cross sectional study (June 2016 to June 2017), we studied the isolation and molecular characterization of multi-drug resistant Escherichia coli (MDR-E. coli) from children suffering from diarrhea. For this purpose, a total of 100 fecal samples were collected with the consent of the parents/ guardians on a prescribed form. The bacterial isolation was done by employing conventional and standard microbiological procedures. Subsequently, all the isolates were identified on the basis of biochemical tests and were further characterized by amplification of 16S rRNA gene followed by di-deoxy sequencing of the amplified product. Afterwards, the isolates were subjected to antimicrobial susceptibility profiling using Kirby Bauer disc diffusion method. A total of 87 E. coli isolates were identified in the current study and majority of the isolates were found sensitive to all or few antimicrobials. However, 14 E. coli isolates were found resistance to multiple drugs including amoxicillin-clavulanic acid, ciprofloxacin, gentamicin, cefoperazone and ofloxacin, hence termed as MDR-E. coli. All of the 14 isolates were further analyzed for the identification of blaCTX-M and blaTEM genes through PCR using specific primers. This resistant was found to be associated with the presence of plasmid encoded beta lactamases genes including blaCTX-M (13/14 E. coli isolates) and blaTEM (9/14 E. coli isolates). Altogether, it was found that ESBLs harboring E. coli is potential source of diarrhea among pediatric diarrheal patients. Therefore, molecular identification and characterization of bacterial pathogens along with antimicrobial susceptibility are critical to understand MDR- E. coli infections.

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.

Pathological and clinical investigations of an outbreak of Blackleg disease due to C. chauvoei in cattle in Punjab, Pakistan.

INTRODUCTION: Clostridium chauvoei (C. chauvoei) is an anaerobic, histotoxic Gram-positive, bacterium causing fatal myonecrosis in livestock with high mortalities. The disease is common in dairy animals, but little is known about the pathophysiology of the disease in exotic (non-native) animals kept under local conditions in Pakistan. METHODOLOGY: Diagnosis of blackleg was made based on hematological and serum biochemical analysis, PCR, necropsy and histopathology. RESULTS: Clinically sick animals exhibited fever, lameness, subcutaneous gaseous swelling and edema particularly in hindquarter and front legs. Hematological analysis showed increases in erythrocyte sedimentation rate and reduces in number of red blood cells, packed cell volume, leukocytes and differential leukocyte count. Serum aspartate aminotransferase, lactate dehydrogenase, alkaline phosphates, alanine aminotransferase, urea, creatinine, creatine kinase, and creatinine phosphokinase were significantly (P < 0.05) higher in the infected animals. At necropsy, swelling areas contained straw-colored fluid with gas bubbles. The muscles were swollen, dark to black and exhibited crepitation sounds at the time of incisions with a rancid odor. Severe pulmonary edema, myocarditis along with petechial hemorrhages, as well as enlargement and congestion of liver and spleen have been observed. Microscopic examination revealed severe inflammatory reaction, edema, and disruption of the myofibrils. Examination of heart, spleen, liver, kidneys, intestine, and lungs showed congestion, severe inflammatory changes with neutrophilic infiltration and necrosis accompanied by dissociation of the normal tissue structure. PCR confirmed C. chauvoei in exudates and different samples of muscles. CONCLUSION: The pathophysiology should be considered in diagnosis of blackleg. The disease is exist in the non-native cattle farms and biosecurity measures have to be elevated.