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Staphylococcus aureus is one of the major pathogens responsible for causing food poisoning worldwide. The emergence of antibiotic resistance in this bacterium is influenced by various factors. Among them, bacterial acquired defense systems described as clustered regularly interspaced short palindromic repeats (CRISPR)-cas system might be involved in antibiotic resistance development in bacteria. The current study was designed to assess the prevalence of S. aureus and its antibiotic resistance profile and identify the relationship of the CRISPR-cas system with antimicrobial resistance, followed by phylogenetic analysis. Total samples (n = 188) of poultry meat were collected from the poultry bird market of Lahore, Punjab, Pakistan. We used both phenotypic (antibiotic disc diffusion) and genotypic methods (PCR) to identify multi-drug resistant (MDR) strains of S. aureus. Additionally, the role of the CRISPR-Cas system in the isolated MDR S. aureus was also assessed. In addition, real-time quantitative PCR (qRT-PCR) was used to evaluate the association of the CRISPR-cas system with antimicrobial resistance. All of the S. aureus isolates showed 100% resistance against erythromycin, 97.5% were resistant to tetracycline, and 75% were resistant to methicillin. Eleven isolates were MDR in the current study. The CRISPR system was found in all MDR isolates, and fifteen spacers were identified within the CRISPR locus. Furthermore, MDR S. aureus isolates and the standard strain showed higher expression levels of CRISPR-associated genes. The correlation of said system with MDR isolates points to foreign gene acquisition by horizontal transfer. Current knowledge could be utilized to tackle antibiotic-resistant bacteria, mainly S. aureus. [ABSTRACT FROM AUTHOR]

aima Muzammil,1 Mohsin Khurshid,1 Muhammad Hidayat Rasool,1 Moeed Ahmad,1 Tamoor Hamid Chaudhry,3 Afreenish Amir,3 Muhammad Salman,3 Zulqarnain Baloch,4 Norah A Alturki,5 Ahmad Alzamami61Department of Microbiology, Government College University Faisalabad, Faisalabad, Pakistan; 2Department of Bioinformatics & Biotechnology, Government College University Faisalabad, Faisalabad, Pakistan; 3Public Health Laboratories Division, National Institute of Health, Islamabad, Pakistan; 4Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming, People's Republic of China; 5Clinical Laboratory Science Department, College of Applied Medical Science, King Saud University, Riyadh, Saudi Arabia; 6Clinical Laboratory Science Department, College of Applied Medical Science, Shaqra University, AlQuwayiyah, Saudi ArabiaCorrespondence: Bilal Aslam, Department of Microbiology, Government College University Faisalabad, Faisalabad, Pakistan, Email [email protected] Ahmad Alzamami, Clinical Laboratory Science Department, College of Applied Medical Science, Shaqra University, AlQuwayiyah, Saudi Arabia, Email [email protected] 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. [ABSTRACT FROM AUTHOR]

Urinary tract infections (UTIs) are a major burden to the health care as it is estimated that around 150 million UTIs occur yearly worldwide. Enterobacteriaceae are the most common agent causing serious urinary tract infections; and multi drug resistent (MDR) cases are increasing day by day. Objectives: To determine the frequency and patient demographics of multidrug resistant urinary tract isolates of Escherichia Coli in a Tertiary Care Hospital. Study Design: Retrospective cross sectional study. Setting: Department of Medicine, Madinah Teaching Hospital / The University of Faisalabad, Faisalabad. Period: May 2016 to Sep 2018. Material & Methods: 187 patients of age 15 to 90 years with positive E. coli on urine culture and sensitivity were included in this retrospective cross sectional study. All those patients with history of dysuria (pain during urination) or frequent urination (more than 7 times per day) were advised urine complete analysis and those with >5 WBCs or pus cells /HPF or having positive for leukocyte esterase and/or nitrite, were advised urine culture and sensitivity. Main outcome variable was the frequency of MDR cases among the culture positive E. coli UTI patients. Results: Among all the cases of E. coli UTI, frequency of MDRE UTI was 66.8% (n=125) and rest 33.2% (n= 62) cases were not MDR UTI. 97.3% patients were resistant to lactam antibiotics, 95.7% were resistant to quinolones and 68.4% were resistant to aminoglycosides. Conclusion: With the rise in the microbe’s resistance to multiple drugs, new therapeutic strategies are to be designed after analyzing our population’s susceptibility pattern. It is not less than a challenge for physicians of a developing nation like Pakistan to treat MDR E.coli UTI cases while taking care of the cost of treatment and controlling the disease burden at the same time. [ABSTRACT FROM AUTHOR]

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 , bla CTX-M-15 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. bla TEM-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.

Antibiotic resistance, which is emerging among pathogenic bacteria, is a leading cause of treatment failure worldwide with particular reference to developing countries. Thus, there is a need to explore and discover novel antimicrobial agents. The objective of this study was to investigate antibacterial activity of spider web metabolites and characterize certain antibacterial compounds. Web solvent extract (methanol, ethanol and acetone) was biologically screened for antibacterial activity against seven multiple drug resistant bacteria including three Gram negative bacteria (E. coli, A. baumannii, and S. typhi) and four gram positive bacteria (S. aureus, Bacillus, E. faecalis and S. pneumonae) using well diffusion test. Solvent extracts with the concentrations of (3.3 mg/ml 4.95 mg/ml 6.6 mg/ml 8.25 mg/ml 9.9 mg/ml and 11.55 mg/ml) were used to check the efficiency against MDR bacteria. Solvent extract of web metabolites exhibited significant activity against all seven MDR bacteria, by producing clear growth inhibition zones ranging in diameter from 04 to 22 mm. Considerable activities was exhibited against E. faecalis (22 mm), E. coli (14 mm) and S. typhi (15 mm). Acetone solvent extracts showed high antibacterial activity against most MDR bacteria. Solvent extracts with concentrations less than 5 mg/ml did not exhibit sufficient activity. Higher activities were related to concentrations higher than 6 mg/ml. Findings of the current study indicated that spider web metabolites may show promising antimicrobial activity against MDR bacteria. Additionally, the results suggest that spider web may be a broad spectrum antimicrobial agent, wherein its individual constituents contribute to its antimicrobial activity. [ABSTRACT FROM AUTHOR]

Objectives: The primary objective of this study is to determine the frequency of rpoB gene mutations within 81-bp RRDR in isolates from Quetta, Pakistan using GeneXpert® MTB/RIF assay. Background: Multi drug resistance is one of the major obstacles in the control of tuberculosis throughout the globe. Study Design: A cross sectional experimental study was designed. Setting: A total of 500 clinical specimens obtained from suspected TB patients at Provincial TB Reference Laboratory, Fatima Jinnah General and Chest Hospital Quetta, Pakistan Period: From January to July 2017. Methodology: Were analyzed by GeneXpert® MTB/RIF assay. Statistical analysis of the data was performed using SPSS version 20. Results: Out of total 500 samples, MTB was detected in 211 (42%) cases by GeneXpert® MTB/RIF assay [positive pulmonary cases 48.8% (206/422) and extra-pulmonary 6.4% (5/78)]. Among 211 MTB positive cases, the assay detected 11 (5.2%) cases with RIF-resistance caused by various rpoB gene mutations within 81-bp RRDR. All the eleven RIF-resistant isolates were found to have mutations only in Probe E and none of cases had RIF-resistance associated with probes A, B, C, and D. Out of 11 RRD cases, 4 (2%) were males and 7 (3.3%) were females. New TB cases were 3 (1.4%) and previously treated cases were 8 (3.8%). Conclusion: In our settings 11/211 (5.2%) of the TB patients showed rifampicin resistance. Probe E mutations (also called codons 531 and 533) were the only rpoB gene mutations detected by GeneXpert® MTB/RIF assay. No mutations were detected in the codons 511, 513, 516, 518, 522 and 526 sequences. [ABSTRACT FROM AUTHOR]

Though emergence of multi-drug resistant bacteria in the environment is a demonstrated worldwide phenomenon, limited research is reported about the prevalence of resistant bacteria in fecal ecology of neonatal calf diarrhea (NCD) animals in Pakistan. The present study aimed to identify and assess the prevalence of bacterial pathogens and their resistance potential in the fecal ecology of NCD diseased animals of Pakistan. The presence of antibiotic resistance genes (bla TEM , bla NDM-1 , bla CTX-M , qnrS) was also investigated. A total of 51 bacterial isolates were recovered from feces of young diarrheic animals (n = 11), collected from 7 cities of Pakistan and identified on the basis of 16S rRNA gene sequence and phylogenetic analysis. Selected isolates were subjected to antimicrobial susceptibility by disc diffusion method while polymerase chain reaction (PCR) was used to characterize the bla TEM , bla NDM-1 , bla CTX-M , qnrS and mcr-1 antibiotic resistance genes. Based on the 16S rRNA gene sequences (Accession numbers: LC488898 to LC488948), all isolates were identified that belonged to seventeen genera with the highest prevalence rate for phylum Proteobacteria and genus Bacillus (23%). Antibiotic susceptibility explained the prevalence of resistance in isolates ciprofloxacin (100%), ampicillin (100%), sulfamethoxazole-trimethoprim (85%), tetracycline (75%), amoxicillin (55%), ofloxacin (50%), ceftazidime (45%), amoxicillin/clavulanic acid (45%), levofloxacin (30%), cefpodoxime (25%), cefotaxime (25%), cefotaxime/clavulanic acid (20%), and imipenem (10%). MICs demonstrated that almost 90% isolates were multi-drug resistant (against at least three antibiotics), specially against ciprofloxacin, and tetracycline with the highest resistance levels for Shigella sp. (NCCP-421) (MIC-CIP up to 75 μg mL -1 ) and Escherichia sp. (NCCP-432) (MIC-TET up to 250 μg mL -1 ). PCR-assisted detection of antibiotic resistance genes showed that 54% isolates were positive for bla TEM gene, 7% isolates were positive for bla CTX-M gene, 23% isolates were positive for each of qnrS and mcr-1 genes, 23% isolates were co-positive in combinations of qnrS and mcr-1 genes and bla TEM and mcr-1 genes, whereas none of the isolate showed presence of bla NDM-1 gene., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Abstract Sequence type 38 is considered a uropathogenic Escherichia coli /enteroaggregative E. coli hybrid associated with multidrug resistance and urinary tract infections. The draft genome sequence of UEC59 from a woman in Pakistan revealed a 5 324 938 bp genome with 5386 coding sequences (CDS), 86 transfer RNA genes and multiple antibiotic resistance genes (bla TEM-1 , CMY-2, sul1, sul2, dfrA17, tetA, mphA) and mobile elements (int1, two transposons, 30 insertion sequence elements, one integrative conjugative element, four plasmids, five prophages), along with many virulence genes. [ABSTRACT FROM AUTHOR]

Background: Pakistan is one of the endemic regions for typhoid fever and paratyphoid fever. This study aimed to identify the evolving antimicrobial sensitivity patterns of Salmonella species causing enteric fever and its implications on the clinical prescribing of antimicrobials., Methods: This was a retrospective descriptive study conducted at a university hospital. Antimicrobial resistance was defined in terms of non-resistant, multidrug resistant (MDR) and extended drug resistant (XDR) as per WHO guidance. Data were collected from the years 2009 and 2019. Chi squared was applied to test for statistical significance (p < 0.05)., Results: A total of 200 patients (100 from 2009 and 100 from 2019) were included in the study. Non-resistant enteric fever cases reduced from 100% in 2009 to 44% in 2019, whereas the MDR and XDR enteric fever cases increased to 16% and 40%, respectively (p < 0.05). Cross tabulation carried out for individual drugs showed an independent rise in the sensitivities of individual first-line antimicrobials., Conclusion: Antimicrobial resistant enteric fever has become a big challenge for Pakistan. The choice of antibiotic prescription has narrowed down to broader spectrum antimicrobials making it difficult to treat, leading to increased morbidity and mortality., Competing Interests: No financial and competing interests to declare

Background: Escherichia coli lineage ST131 predominates across various spectra of extra-intestinal infections, including urinary tract infection (UTI). The distinctive resistance profile, diverse armamentarium of virulence factors and rapid global dissemination of ST131 E. coli makes it an intriguing pathogen. However, not much is known about the prevalence and genetic attributes of ST131 lineage in Pakistan., Methods: We estimated prevalence and genetic attributes of E. coli ST131 isolates causing UTI among 155 randomly selected samples. Samples were analyzed for phylogenetic grouping, O-typing and fumC/fimH typing. Isolates were further tested for the ESBL and virulence factors using PCR., Results: Overall, 59% of the UPEC isolates belonged to the phylogenetic group B2, followed by D = 28%, B1 = 8% and A = 5%. Among 18 different Sequence-types, ST131 was the dominant lineage (n = 71; 46%) out of which 72% of the isolates were assigned to the phylogenetic group B2, while 61% adhered to the serogroup O25b. FumC/fimH typing confirmed 49% of the ST131 as H30 sub-types. In this study, significant numbers of the identified ST131 isolates were MDR and 42% showed ESBL phenotypes, out of which 37% carried bla- CTX-M-15 . Moreover, different virulence factors were detected in following percentages: fimH,155(100%), iutA 86 (55%), feoB 76 (49%), papC 75 (48%), papGII 70 (45%), kpsMTII 40 (26%), papEF 37 (24%), fyuA 37 (24%), usp 22 (14%), papA 20 (13%), sfa/foc20 (13%), hlyA 18 (12%), afa 15 (10%), cdtB 11 (7%), papGI 6 (4%), papGIII 6 (4%), kpsMTIII 4 (3%) and bmaE2 (1%)., Conclusion: Conclusively, this study provides important insight into the genetic and virulence attributes of pandemic MDR ST131 strains involved in UTIs. It also highlights higher prevalence of ST131-O25b-H30 UPEC isolates in patients, which was previously unreported from this part of globe.

Background: South-Asia is known as a hub for multidrug-resistant (MDR) bacteria. Unfortunately, proper surveillance and documentation of MDR pathogens is lacking in Pakistan. The alarming increase in the prevalence of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae is a serious problem. From this perspective, we analysed published data regarding ESBL-producing Enterobacteriaceae in different regions of Pakistan. Methods: A meta-analysis was performed to determine the prevalence of ESBL-producing Enterobacteriaceae in Pakistan. A Web-based search was conducted in electronic databases, including PubMed, Scopus and PakMedi Net (for non-indexed Pakistani journals). Articles published (in either indexed or non-indexed journals) between January 2002 and July 2016 were included in the study. Relevant data were extracted, and statistical analysis was performed using the Metaprop command of STATA version 14.1. Results: A total of 68 studies were identified from the electronic data base search, and 55 of these studies met our inclusion criteria. Pakistan’s overall pooled proportion of ESBL-producers was 0.40 (95% CI: 0.34–0.47). The overall heterogeneity was significant (I2 = 99.75%, p < 0.001), and significant ES = 0 (Z = 18.41, p < 0.001) was found. OXA, SHV, TEM and CTX-M were the most commonly found gene variants for ESBLs in these studies. Conclusion: The prevalence of ESBL-producing Enterobacteriaceae is high in Pakistan. Little is known about the annual frequency of ESBLs and their prevalence in different provinces of Pakistan. No data are available regarding ESBL frequency in Baluchistan. This underscores an urgent demand for regular surveillance to address this antimicrobial resistance problem. Surveillance to better understand the annual ESBL burden is crucial to improve national and regional guidelines. [ABSTRACT FROM AUTHOR]

Aims: Pyrazinamide (PZA) is an important component of first-line tuberculosis (TB) treatment because of its distinctive capability to kill subpopulations of persister Mycobacterium tuberculosis (MTB). The significance of PZA can be understood by its inclusion in the most recent World Health Organization-recommended multidrug-resistant (MDR) TB regimen. Very little information is available about the prevalence of PZA-resistant TB from geographically distinct regions of high burden countries, including Khyber Pakhtunkhwa (KPK), Pakistan, because drug susceptibility testing (DST) of PZA is not regularly performed due to the complexity. In this study, we aimed to find the prevalence of PZA resistance in geographically distinct, Pashtun-dominant KPK Province of Pakistan and its correlation with other first- and second-line drug resistance., Materials and Methods: In this study, DST of PZA was performed through an automated BACTEC MGIT 960 system (BD Diagnostic Systems). The resistant samples were further subjected to DST of isoniazid (INH), rifampicin (RIF), ethambutol (EMB), streptomycin (SM), moxicillin (MOX), amikacin (AMK), ofloxacin (OFX), kanamycin (KM), and capreomycin (CAP)., Results: Out of 1,075 MTB-positive isolates, 83 (7.7%) were found to be resistant to PZA. Among the PZA-resistant isolates, 76 (90-91.6%) and 67 (80-80.7%) were found to be resistant to INH and RIF, respectively, whereas 63 (76%) were resistant to both first-line drugs, INH and RIF (MDR-TB). The resistance level of EMB, OFX, and SM was also significantly high in PZA resistance, 35 (42%), 40 (48%), and 41 (49-50%) respectively., Conclusion: PZA resistance is significantly associated with other first- and second-line drug resistance. A significant number of PZA-resistant isolates are MDR cases. Therefore, DST of PZA should regularly be performed along with other drugs for better management of treatment of MDR and extensively drug resistant (XDR), to avoid side effects in patients.