Abdul Haque

Detection of Vi-Negative Salmonella enterica Serovar Typhi in the Peripheral Blood of Patients with Typhoid Fever in the Faisalabad Region of Pakistan

ABSTRACT The synthesis and transportation proteins of the Vi capsular polysaccharide of Salmonella enterica serovar Typhi (serovar Typhi) are encoded by the viaB operon, which resides on a 134-kb pathogenicity island known as SPI-7. In recent years, Vi-negative strains of serovar Typhi have been reported in regions where typhoid fever is endemic. However, because Vi negativity can arise during in vitro passage, the clinical significance of Vi-negative serovar Typhi is not clear. To investigate the loss of Vi expression at the genetic level, 60 stored strains of serovar Typhi from the Faisalabad region of Pakistan were analyzed by PCR for the presence of SPI-7 and two genes essential for Vi production: tviA and tviB. Nine of the sixty strains analyzed (15%) tested negative for both tviA and tviB; only two of these strains lacked SPI-7. In order to investigate whether this phenomenon occurred in vivo, blood samples from patients with the clinical symptoms of typhoid fever were also investigated. Of 48 blood samples tested, 42 tested positive by fliC PCR for serovar Typhi; 4 of these were negative for tviA and tviB. Three of these samples tested positive for SPI-7. These results demonstrate that viaB-negative, SPI-7-positive serovar Typhi is naturally occurring and can be detected by PCR in the peripheral blood of typhoid patients in this region. The method described here can be used to monitor the incidence of Vi-negative serovar Typhi in regions where the Vi vaccine is used.

Multiplex PCR for differential diagnosis of emerging typhoidal pathogens directly from blood samples

Classically Salmonella enterica serovar Typhi (S. Typhi) is associated with typhoid, a major health problem in developing countries. However, in recent years S. Paratyphi A and Vi-negative variants of S. Typhi have emerged rapidly. We have developed a nested multiplex PCR targeting five different genes for differential diagnosis of typhoidal pathogens which has been optimized to be directly applicable on clinical blood samples. Of 42 multiplex PCR-positive blood samples, 26, nine, and two were Vi-positive S. Typhi, Vi-negative S. Typhi and S. Paratyphi A, respectively, and five patients were found to have mixed infection. Seventeen patients grew Salmonella from blood culture and the remaining 25 were positive in the Salmonella-specific PCR. Tests with several common pathogens confirmed the specificity of the assay. We conclude that the proposed multiplex PCR is rapid, sensitive and specific for the diagnosis of typhoidal pathogens directly from blood samples.

Molecular profiling of antimicrobial resistance and integron association of multidrug-resistant clinical isolates of Shigella species from Faisalabad, Pakistan

Bacillary dysentery, common in developing countries, is usually caused by Shigella species. A major problem in shigellosis is the rapid emergence of multidrug-resistant strains. This is the first detailed molecular study on drug resistance of Shigella isolates from the Faisalabad region of Pakistan. Ninety-five Shigella isolates obtained after screening of 2500 stool samples were evaluated for in vitro resistance to commonly used antimicrobial agents; the presence or absence of 20 of the most relevant drug resistance genes; and the prevalence of integrons 1, 2, and 3. Shigella flexneri was found to be the most prevalent and most resistant species. Collectively, high resistance was found towards ampicillin (96.84%), tetracycline (93.68%), streptomycin (77.89%), and chloramphenicol (72.63%). Significant emerging resistance was detected towards the modern frontline drugs ciprofloxacin (12.63%), cefradine (17.89%), ceftriaxone (20.00%), cefoperazone (22.10%), and cefixime (28.42%). Prevalence rates for bla(TEM), bla(CTX-M), gyrA, gyrB, qnrS, aadA1, strAB, tetA, tetB, catA, and catP were 78.94%, 12.63%, 20.00%, 21.05%, 21.05%, 67.36%, 42.10%, 12.63%, 53.68%, 33.68%, and 25.26%, respectively. Class 2 integrons (42.10%) were more common in the local isolates. Simultaneous detection of class 1 and 2 integrons in some isolates and a rapidly emerging resistance to modern frontline drugs are the major findings of this study.

Effects of ampicillin, gentamicin, and cefotaxime on the release of Shiga toxins from Shiga toxin-producing Escherichia coli isolated during a diarrhea episode in Faisalabad, Pakistan.

The Shiga toxin-producing Escherichia coli (STEC) is an emerging foodborne pathogen. The proportion of cases attributed to STEC in an episode of diarrhea in the Faisalabad region of Pakistan was investigated. In addition, as increase in Shiga toxin (Stx) release after exposure to various antimicrobial agents is widely reported, we also elucidated the in vitro effects of three commonly used antibiotics (ampicillin, gentamicin, and cefotaxime) on Stx release. Isolation and detection of STEC was done using enzyme-linked immunosorbent assay and polymerase chain reaction, followed by phenotypic characterization. In vitro Stx release from isolated STEC was determined using enzyme-linked immunosorbent assay, and Stx-induced verocytotoxicity was quantified using cytotoxicity detection assay. STEC was detected in 5 (21.7%) of 23 patients. Exposure to minimum inhibitory concentration (MIC) of ampicillin, gentamicin, and cefotaxime resulted in a considerable decrease in toxin release and level of cytotoxicity in most of the STEC isolates when compared with control (without antibiotic exposure). Exposure to sub-MIC of ampicillin resulted in a relative increase in Stx release and cytotoxicity (p <or= 0.05) in three of the four isolates tested, whereas a decreasing trend was observed in isolates exposed to sub-MICs of gentamicin and cefotaxime. Sub-MIC of gentamicin resulted in largest decrease in Stx release and a similar trend was observed with cefotaxime to a lesser extent. In conclusion, these in vitro observations suggested that sub-MIC of ampicillin may stimulate Stx release and level of cytotoxicity and therefore should be avoided. Gentamicin did not show such effects and therefore may be considered for STEC antimicrobial therapy.

Virulence profile of different phylogenetic groups of locally isolated community acquired uropathogenic E. coli from Faisalabad region of Pakistan.

BackgroundUropathogenic E.coli (UPEC) are among major pathogens causing urinary tract infections. Virulence factors are mainly responsible for the severity of these emerging infections. This study was planned to investigate the distribution of virulence genes and cytotoxic effects of UPEC isolates with reference to phylogenetic groups (B2, B1, D and A) to understand the presence and impact of virulence factors in the severity of infection in Faisalabad region of Pakistan.MethodsIn this study phylogenetic analysis, virulence gene identification and cytotoxicity of 59 uropathogenic E.coli isolates obtained from non-hospitalized patients was studied.ResultsAmong 59 isolates, phylogenetic group B2 (50%) was most dominant followed by groups A, B1 (19% each) and D (12%). Isolates present in group D showed highest presence of virulence genes. The prevalence hlyA (37%) was highest followed by sfaDE (27%), papC (24%), cnf1 (20%), eaeA (19%) and afaBC3 (14%). Highly hemolytic and highly verotoxic isolates mainly belonged to group D and B2. We also found two isolates with simultaneous presence of three fimbrial adhesin genes present on pap, afa, and sfa operons. This has not been reported before and underlines the dynamic nature of these UPEC isolates.ConclusionsIt was concluded that in local UPEC isolates from non-hospitalized patients, group B2 was more prevalent. However, group D isolates were most versatile as all were equipped with virulence genes and showed highest level of cytotoxicity.

Preparation and evaluation of immunogenic conjugates of Salmonella enterica serovar Typhi O-specific polysaccharides with diphtheria toxoid

Typhoid fever, caused by Salmonella enterica serovar Typhi (S. Typhi), is a major health problem particularly in developing countries. The available vaccines have certain limitations regarding their efficacy, and inability to induce an immune response especially in individuals under 2 years of age. Conjugate vaccines which consist of a bacteria-specific polysaccharide chemically bound to a carrier protein overcome these problems by inducing a T-cell dependent immune response characterized by enhanced immunogenicity in all ages. In this study, O-specific polysaccharides (OSP) of S. Typhi were conjugated to diphtheria toxoid (DT) using adipic acid dihydrazide (ADH) as a linker. These conjugates (OSP-AH-DT) were then evaluated for their immunogenicity using mice as a model and showed significantly higher levels of IgG ELISA titers (P = 0.0241 and 0.0245) than lipopolysaccharides alone. Different immunization schedules were compared and it was found that schedule-B (three injections with 4-weeks interval) induced higher immune responses than schedule-A (three injections with 2-weeks interval). We showed that diphtheria toxoid can be successfully employed as a carrier protein for conjugation with Salmonella OSP and play an important role in facilitating adequate immune response.

Multiple drug resistance patterns in various phylogenetic groups of uropathogenic E.coli isolated from Faisalabad region of Pakistan

The objective of this work was the phylogenetic characterization of local clinical isolates of uropathogenic E. coli with respect to drug resistance. A total of 59 uropathogenic E. coli responsible for community acquired urinary tract infections were included in this study. A triplex PCR was employed to segregate each isolate into four different phylogenetic groups (A, B1, B2 and D). Drug resistance was evaluated by disc diffusion method. The drugs used were ampicillin, aztreonam, cefixime, cefoperazone, ceftriaxone, cephradine among β-lactam group; amikacin, gentamicin, and streptomycin among aminoglycosides; nalidixic acid and ciprofloxacin from quinolones; trimethoprim-sulfomethoxazole, and tetracycline. Among 59 uropathogenic E. coli isolates majority belonged to phylogenetic group B2 (50%) where as 19% each belonged to groups A and B1, and 12% to group D. All the isolates were multiple drug resistant (MDR). Most effective drugs against Group A, B1, and B2 were gentamicin, amikacin and cefixime; ceftriaxone and quinolones; and ceftriaxone and amikacin, respectively. Group D isolates were found to be highly resistant to all drugs. Our results have shown emergence of MDR isolates among uropathogenic E. coli with dominance of phylogenetic group B2. However, it was found that group D isolates were though less frequent, more drug resistant as compared with group B2. Groups A and B1 were relatively uncommon. Amikacin, ceftriaxone and gentamicin were the most effective drugs in general.

Effect of biofilm formation on the excretion of Salmonella enterica serovar Typhi in feces

OBJECTIVES We hypothesized that Salmonella enterica serovar Typhi (S. Typhi) with higher biofilm and capsule production capability are more able to survive continuously in typhoid patients/carriers, with subsequent prolonged shedding in feces. METHODS Bacterial cell release from biofilm (produced in vitro and confirmed by specific staining and electron microscopy) and comparative cytotoxicity were studied on Caco2 cells. Functionality of the biofilm diffusion barrier was tested against ciprofloxacin. Biofilm production was graded and semi-quantified as -, +, ++, +++, and ++++. RESULTS Out of 30 isolates, 23 produced biofilm. The average post-treatment detection of S. Typhi in blood was 7-13 days and in stool was 13-32 days. A fall in cell count from 10⁴ to approximately 10¹ over the course of 3 days as compared to total elimination of planktonic cells in 16 h after ciprofloxacin application substantiated the protective role of biofilm. Lactic dehydrogenase release ranged from 38% in non-biofilm producers to 97% in the highest biofilm producers, indicating increased pathogenic behavior. CONCLUSIONS The period of S. Typhi clearance from typhoid patients after recovery was found to be directly related to biofilm production capability.

Characterization of putative multidrug resistance transporters of the major facilitator-superfamily expressed in Salmonella Typhi

Multidrug resistance mediated by efflux pumps is a well-known phenomenon in infectious bacteria. Although much work has been carried out to characterize multidrug efflux pumps in Gram-negative and Gram-positive bacteria, such information is still lacking for many deadly pathogens. The aim of this study was to gain insight into the substrate specificity of previously uncharacterized transporters of Salmonella Typhi to identify their role in the development of multidrug resistance. S. Typhi genes encoding putative members of the major facilitator superfamily were cloned and expressed in the drug-hypersensitive Escherichia coli strain KAM42, and tested for transport of 25 antibacterial compounds, including representative antibiotics of various classes, antiseptics, dyes and detergents. Of the 15 tested putative transporters, STY0901, STY2458 and STY4874 exhibited a drug-resistance phenotype. Among these, STY4874 conferred resistance to at least ten of the tested antimicrobials: ciprofloxacin, norfloxacin, levofloxacin, kanamycin, streptomycin, gentamycin, nalidixic acid, chloramphenicol, ethidium bromide, and acriflavine, including fluoroquinolone antibiotics, which were drugs of choice to treat S. Typhi infections. Cell-based functional studies using ethidium bromide and acriflavine showed that STY4874 functions as a H(+)-dependent exporter. These results suggest that STY4874 may be an important drug target, which can now be tested by studying the susceptibility of a STY4874-deficient S. Typhi strain to antimicrobials.

Genotyping of Rotaviruses Detected in Children Admitted to Hospital From Faisalabad Region, Pakistan

Rotavirus infection is very common in developing countries and occurs at least once in children under the age of 5 years. The rate of detection of rotavirus infection in various age groups (0–5 years) in patients with gastroenteritis admitted to hospital from the Faisalabad region, Pakistan is reported. Out of 300 fecal samples, 189 (57.3%) were positive for rotavirus by immunoassay. Patients aged 7–12 months (35.4%) were infected most commonly followed by the age group 0–6 months (28%). Different genotypes of rotavirus were identified by hemi‐nested RT‐PCR. The most common genotype was G1P[8] (25.3%), followed by G1P[6] (21.1%). Other genotypes were G1P[9], G2P[6], G9P[10]), G3P[8] (1.5%), and G9P[11] (1%). There were two (1%) cases of mixed G genotype, one patient with two genotypes G1, G10 and another patient with 3 genotypes G1, G10, and G12. There were 6 (3.1%) cases of mixed P genotypes, 3 P[4], P[11] and 3 P[8], P[11]. These results provide an outline of rotavirus infection in this area for the first time. J. Med. Virol. 84:2003–2007, 2012.