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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 fum C/ fim H 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. Fum C/ fim H 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: fim H,155(100%), iut A 86 (55%), feo B 76 (49%), pap C 75 (48%), papGII  70 (45%), kpsMTII 40 (26%), pap EF 37 (24%), fyu A 37 (24%), usp 22 (14%), pap A 20 (13%), sfa/foc 20 (13%),  hly A 18 (12%), afa 15 (10%),  cdt B 11 (7%), papGI 6 (4%), papGIII 6 (4%),  kpsMTIII 4 (3%) and bma E2 (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.

The emergence of carbapenem-resistant and hypervirulent hypermucoviscous strains poses a significant public health challenge. We determined the MDR profiles, antibiotic resistance factors, virulence gene complement, and hypermucoviscous features of 200 clinical isolates from two major tertiary care hospitals in Islamabad and Rawalpindi, Pakistan. Susceptibility profiling and phenotypic analysis were performed according to the CLSI guidelines. Genetic determinants of antibiotic resistance and virulence were detected by PCR. Biofilm formation analysis was performed by microtiter plate assay. The isolates displayed a high degree of antibiotic resistance: 36% MDR-CRKP; 38% carbapenem resistance; 55% gentamicin resistance; 53% ciprofloxacin resistance; and 59% aztreonam resistance. In particular, the level of resistance against fosfomycin (22%) and colistin (15%) is consistent with previous reports of increased resistance levels. Combined resistance to carbapenem and colistin was 7%. Genetic factors associated with colistin resistance ( and genes) were detected in 12 and 9% of the isolates, respectively. Significant differences in resistance to gentamicin and levofloxacin were observed between the 200 isolates. Many of the isolates harbored genes specifying extended-spectrum and/or carbapenem-resistant β-lactamases: (46%), (39%), and (24%). The prevalence of the hypermucoviscous phenotype was 22% and 13% of the MDR isolates carried the gene (regulator for mucoid phenotype). Key virulence factor genes detected include those encoding: porins ( and ; at 56 and 55% prevalence, respectively); adhesins ( and ; at 19, 18, and 22% prevalence, respectively); and the polysaccharide regulator, , at 16% prevalence. This report highlights carbapenem-resistant (CRKP) prevalence, emerging resistance to fosfomycin, and the presence of and in colistin-resistant isolates. Further, the detection of signifies the prevalence of the hypermucoviscous trait in CRKP clinical isolates from Pakistan.

Antibiotic use has risen sharply in recent decades, fueling resistance. Since China reported colistin resistance in 2015, global concern has grown. While few cases of colistin resistance have been reported in Enterobacteriaceae from Pakistan, molecular factors remain unclear. This study aimed to determine the prevalence of colistin-resistant Enterobacteriaceae (ColRE) in a tertiary care hospital in Abbottabad, Pakistan. Altogether 89 Enterobacteriaceae isolates were collected from in-patients or out-patients at Ayub Teaching Hospital Abbottabad. The isolates were identified using API 20E kit and antibiotic susceptibility testing was performed following CLSI guidelines. Molecular screening of antibiotic resistance genes was conducted by PCR. The results showed E. coli (40.4%) as the most common pathogen, followed by K. pneumoniae (21.3%), S. typhi (19%), Enterobacter (9%), Raoultella ornithinolytica (4.4%), K. aerogenes (2.2%), Citrobacter (2.2%) and K. oxytoca (1%). Antibiotic testing showed highest resistance to aztreonam and cefepime (65.1%), and lowest to colistin (29.2%) and meropenem (22.4%). Overall, 48.3% of the isolates were multidrug resistant (MDR), with K. pneumoniae showing the highest MDR rate (68.4%). Molecular screening of colistin resistance genes found 15.3% of isolates positive for mcr-1 or mcr-2 genes. Simultaneous presence of mcr-1 and mcr-2 was identified in 3.8% of isolates. The study also found the first case of K. aerogenes with mcr-1 positivity. In conclusion, the study reveals a high rate of colistin resistance Enterobacteriaceae with notable presence of plasmid mediated genes. The study recommends nationwide surveillance of ColRE, with emphasis on the screening of plasmid-mediated determinants.

Community-acquired urinary tract infections are associated with significant morbidity, and uropathogenic (UPEC) alone causes 90% of urinary tract infections. This bacterium retains a diverse armament of virulence factors including fimbria, hemolysins, and siderophores production. In a post invasion scenario, formation of intracellular communities mimic biofilm-like characteristics and are linked to recurrent urinary tract infections. We investigated the effects of different frontline antibiotics on the formation, inhibition, and eradication of biofilms of virulent UPEC strains. A total of 155 UPEC strains were scrutinized for various virulence factors including gelatinase, cell surface hydrophobicity, hemagglutination, and serum bactericidal activity. Biofilm formation was confirmed by three different methods: Congo red agar, test tube, and tissue culture plate method. Biofilm inhibition and eradication assays were performed according to the standard protocols. Topographical analysis of biofilms was done by scanning electronic microscopy (SEM). Out of 155 strains, 113 (73%) were strong biofilm formesr, while 37 (24%) produced biofilms at moderate level. Significant differences were observed between MICs of planktonic cells (MIC-p) and MICs of UPEC biofilms (MIC-b). Among tested frontline antibiotics, levofloxacin successfully inhibited biofilms at a concentration of 32 µg/mL, while trimethoprim eradicated biofilms at higher concentrations (512-1024 µg/mL). Ciprofloxacin treatment at sub-MIC level significantly enhanced biofilm formation (P<0.05). The majority of UPEC strains are strong biofilm formers and show higher tolerance towards frontline antibiotics in biofilm form. We observed significant inhibitory effects of levofloxacin (32 µg/mL) on UPEC biofilms, while treatment with sub-minimal concentrations of ciprofloxacin significantly enhanced biofilm formation. Out of all tested antibiotics, trimethoprim (512-1024 µg/mL) eradicated UPEC biofilms.

Background: Pseudomonas aeruginosa is an opportunistic pathogen involved in number of hospital-acquired infections such as catheter-associated urinary tract infections, bacteremia, septicemia, skin infections, and ventilator-associated pneumoniae. Biofilm formation is an important trait implicated in chronic infections, such as cystic fibrosis and chronic pulmonary obstruction. We evaluated effects of gentamicin, cefepime, and ciprofloxacin on biofilm of P. aeruginosa. Materials and Methods: A total of 266 isolates were collected from the Armed Forces Institute of Pathology (AFIP). Antibiotic susceptibility was assessed by double disk synergy testing. ESBL and carbapenemase detection was performed by phenotypic testing. Molecular screening of the genes was done by PCR. Micro-dilution broth method was used to determine minimum inhibitory concentrations of antibiotics. Biofilm formation was done by micro-titer plate assay. Results: Overall, 20% of the P. aeruginosa isolates were extensively drug- resistant (XDR-PA), and 25% were multi- drug-resistant (MDR-PA). Likewise, 43% of the isolates were ESBL producers, and carbapenemase production was detected in 40% of the isolates. Molecular analysis confirmed occurrence of different resistant factors in ESBL- positive isolates; 67% carried [bla.sub.TEM], 62% [bla.sub.CTXM-15], 41% [bla.sub.SHV], 34% [bla.sub.CTXM-14], and 33% [bla.sub.OXA-1]. In addition, 68% of the carbapenem-resistant isolates were positive for [bla.sub.NDM-1], 25% for [bla.sub.OXA-48], and 22% for [bla.sub.KPC-2]. Biofilm formation was assessed for 234 isolates, out of which 28% were strong biofilm formers. Moderate and weak biofilm formers constituted 46% and 23%, respectively. Overall, ciprofloxacin, levofloxacin, and cefepime showed inhibitory effects on P. aeruginosa biofilms. Antibiotics in combination showed strong synergistic effects (ciprofloxacin and cefepime), while gentamicin and cefepime resulted in complete eradication of P. aeruginosa biofilm. Conclusion: We confirm strong synergistic effects of gentamicin and cefepime that completely eradicated P. aeruginosa biofilm. We further confirm inhibitory effects of ciprofloxacin, levofloxacin, and cefepime on P. aeruginosa biofilms. Hence, combination therapy can be more effective against biofilm-associated infections. Keywords: synergistic effects, gentamicin, cefepime, biofilm, Pseudomonas aeruginosa

Background: Widespread use of antibiotics as growth promoters and prophylactic agents has dramatic consequences for the development of antibiotic resistance. In this study, we investigated effects of selected antibiotics on bacterial biofilms and performed extensive antibiotic and VF profiling of poultry-meat associated E. coli strains. Methods: Antibiotic susceptibility was performed by a disc diffusion method, followed by molecular screening of resistance and virulence determinants. Further biofilm formation assays, MIC-p, MIC-b, MBIC and MBEC, were performed using standard tissue culture plate method. Results: In total, 83 (75%) samples were confirmed as E. coli from poultry sources, 26 different antibiotics were tested, and maximum numbers of the isolates were resistant to lincomycin (100%), while the least resistance was seen against cefotaxime (1%) and polymyxin B (1%). Overall, 48% of the isolates were ESBL producers and 40% showed carbapenemase activity; important virulence genes were detected in following percentages: fimH32 (39%), papC21 (25%), iutA34 (41%), kpsMT-II23 (28%), papEF9 (11%), papGII22 (27%) and fyuA13 (16%). Colistin showed remarkable anti-biofilm activity, while at sub-MIC levels, gentamicin, ceftriaxone and enrofloxin significantly (p < 0.01) inhibited the biofilms. A strong induction of bacterial biofilm, after exposure to sub-minimal levels of colistin clearly indicates risk of bacterial overgrowth in a farm environment, while use of colistin aggravates the risk of emergence of colistin resistant Enterobacteriaceae, a highly undesirable public health scenario.

Background: The emergence of carbapenem-resistant and hypervirulent hypermucoviscous Klebsiella pneumoniae strains poses a significant public health challenge. We determined the MDR profiles, antibiotic resistance factors, virulence gene complement, and hypermucoviscous features of 200 clinical K. pneumoniae isolates from two major tertiary care hospitals in Islamabad and Rawalpindi, Pakistan. Methods: Susceptibility profiling and phenotypic analysis were performed according to the CLSI guidelines. Genetic determinants of antibiotic resistance and virulence were detected by PCR. Biofilm formation analysis was performed by microtiter plate assay. Results: The isolates displayed a high degree of antibiotic resistance: 36% MDRCRKP; 38% carbapenem resistance; 55% gentamicin resistance; 53% ciprofloxacin resistance; and 59% aztreonam resistance. In particular, the level of resistance against fosfomycin (22%) and colistin (15%) is consistent with previous reports of increased resistance levels. Combined resistance to carbapenem and colistin was 7%. Genetic factors associated with colistin resistance (mcr-1 and mcr-2 genes) were detected in 12 and 9% of the isolates, respectively. Significant differences in resistance to gentamicin and levofloxacin were observed between the 200 isolates. Many of the isolates harbored genes specifying extended-spectrum and/or carbapenem-resistant P-lactamases: [bla.sub.CTX-M-15] (46%), [bla.sub.NDM-1] (39%), and [bla.sub.OXA-48] (24%). The prevalence of the hypermucoviscous phenotype was 22% and 13% of the MDR isolates carried the rmpA gene (regulator for mucoid phenotype). Key virulence factor genes detected include those encoding: porins (ompK35 and ompK36; at 56 and 55% prevalence, respectively); adhesins (fimH, mrkD, and ycfM; at 19, 18, and 22% prevalence, respectively); and the polysaccharide regulator, bss, at 16% prevalence. Conclusion: This report highlights carbapenem-resistant K. pneumoniae (CRKP) prevalence, emerging resistance to fosfomycin, and the presence of mcr-1 and mcr-2 in colistin-resistant isolates. Further, the detection of rmpA signifies the prevalence of the hypermucoviscous trait in CRKP clinical isolates from Pakistan. Keywords: Klebsiella pneumoniae, multidrug resistance, carbapenemases, colistin resistance, hypermucoviscous K. pneumoniae

Background: Widespread use of antibiotics as growth promoters and prophylactic agents has dramatic consequences for the development of antibiotic resistance. In this study, we investigated effects of selected antibiotics on bacterial biofilms and performed extensive antibiotic and VF profiling of poultry-meat associated E. coli strains. Methods: Antibiotic susceptibility was performed by a disc diffusion method, followed by molecular screening of resistance and virulence determinants. Further biofilm formation assays, MIC-p, MIC-b, MBIC and MBEC, were performed using standard tissue culture plate method. Results: In total, 83 (75%) samples were confirmed as E. coli from poultry sources, 26 different antibiotics were tested, and maximum numbers of the isolates were resistant to lincomycin (100%), while the least resistance was seen against cefotaxime (1%) and polymyxin B (1%). Overall, 48% of the isolates were ESBL producers and 40% showed carbapenemase activity; important virulence genes were detected in following percentages: fimH32 (39%), papC21 (25%), iutA34 (41%), kpsMT-II23 (28%), papEF9 (11%), papGII22 (27%) and fyuA13 (16%). Colistin showed remarkable anti-biofilm activity, while at sub-MIC levels, gentamicin, ceftriaxone and enrofloxin significantly (p< 0.01) inhibited the biofilms. A strong induction of bacterial biofilm, after exposure to sub-minimal levels of colistin clearly indicates risk of bacterial overgrowth in a farm environment, while use of colistin aggravates the risk of emergence of colistin resistant Enterobacteriaceae, a highly undesirable public health scenario.

Antibiotic resistance in urinary tract infections is a major global challenge and improved cost-effective and high throughput antibiotic susceptibility tests (AST) are urgently needed to inform correct antibiotic selection. We evaluated a high throughput microfluidic test strip for AST and minimum inhibitory concentration (MIC) determination in 20 urinary pathogenic E. coli (UPEC) isolates using six commonly prescribed or therapeutically beneficial antibiotics. The microfluidic MIC performs broth microdilution in 1 microliter volume capillaries, 100 X smaller than standard broth microdilution. Each test strip contains 10 parallel capillaries which are dipped into a single well of a 96 well plate, significantly increasing throughput over a microtitre plate. When tested with clinical UPEC isolates at standardised inoculum density, these devices gave 100% essential agreement (+/- 1 doubling dilution of antibiotic) to the gold standard microplate broth microdilution method described by CLSI. Although for some antibiotic/isolate combinations an earlier endpoint readout reduced accuracy, MIC test strips read at a 6h endpoint still gave 69 to 100 % essential agreement depending on the antibiotic. Growth could be detected significantly earlier than 6h, but with a trade-off between speed vs accuracy. These high-throughput, multiplexed test strips could be used to increase throughput and give faster results than microplates while retaining the core broth microdilution methodology of gold standard techniques for AST and MIC determination. Competing Interest Statement ADE is one of the inventors of patent application protecting aspects of the novel microfluidic devices tested in this study and is a director and shareholder in Capillary Film Technology Ltd, a company holding a commercial license to this patent application: WO2016012778. Capillary assay device with internal hydrophilic coating. AD Edwards, NM Reis.