Antimicrobial resistance trends in blood culture positive Salmonella Paratyphi A isolates from Pondicherry, India.

Enteric fever is a public health problem with the upsurge in the occurrence of Salmonella isolates that are resistant to ciprofloxacin. In this study, a total of 284 blood culture isolates of S. Paratyphi A were investigated. Of these isolates, 281 (98.9%) were nalidixic acid resistant. A high rate (6.3%) of high-level resistance (≥ 4 µg/mL) was found to ciprofloxacin. The isolates with ciprofloxacin minimum inhibitory concentrations (MICs) of ≥ 12 µg/mL had 4 mutations, 2 mutations within the quinolone resistance-determining region of gyrA and 2 mutations also in parC. According to the Clinical Laboratory Standards Institute 2012 MIC breakpoints, 75.0% of isolates were resistant to ciprofloxacin. Finally, 3 major pulsed-field gel electrophoresis patterns were observed among the S. Paratyphi A isolates. The spread of fluoroquinolone resistant S. Paratyphi A necessitates a change toward 'evidence-based' treatment for enteric fever. The research provides a perspective on the increasing prevalence of antimicrobial resistant S. Paratyphi A isolates in this region of India.

Early diagnosis of typhoid fever by nested PCR for flagellin gene of Salmonella enterica serotype Typhi.

BACKGROUND & OBJECTIVES: Typhoid fever caused by Salmonella Typhi continues to be a major health problem in spite of the use of antibiotics and the development of newer antibacterial drugs. Inability to make an early laboratory diagnosis and resort to empirical therapy, often lead to increased morbidity and mortality in cases of typhoid fever. This study was aimed to optimize a nested PCR for early diagnosis of typhoid fever and using it as a diagnostic tool in culture negative cases of suspected typhoid fever. METHODS: Eighty patients with clinical diagnosis of typhoid fever and 40 controls were included in the study. The blood samples collected were subjected to culture, Widal and nested PCR targeting the flagellin gene of S. Typhi. RESULTS: The sensitivity of PCR on blood was found to be 100 per cent whereas the specificity was 76.9 per cent. The positive predictive value (PPV) of PCR was calculated to be 76.9 per cent with an accuracy of 86 per cent. None of the 40 control samples gave a positive PCR. INTERPRETATION & CONCLUSIONS: Due to its high sensitivity and specificity nested PCR can be used as a useful tool to diagnose clinically suspected, culture negative cases of typhoid fever.

Antimicrobial resistance trends in blood culture positive Salmonella Typhi isolates from Pondicherry, India, 2005-2009.

Typhoid fever is caused by Salmonella enterica serovar Typhi, a major public health concern in developing countries. Recently, there has been an upsurge in the occurrence of bacterial isolates that are resistant to ciprofloxacin, and the emergence of broad spectrum ß-lactamases in typhoidal salmonellae constitutes a new challenge for the clinician. A total of 337 blood culture isolates of S. Typhi, isolated from Pondicherry, India, between January 2005 and December 2009, were investigated using phenotypic, molecular and serological methods. Of the 337 isolates, 74 (22%) were found to be multidrug resistant (MDR) and 264 (78%) nalidixic acid resistant (NAR). Isolates with reduced susceptibility to ciprofloxacin possessed single mutations in the gyrA gene. A high rate of resistance (8%) was found to ciprofloxacin. All isolates with a ciprofloxacin MIC ≥ 4 mg/L possessed both double mutations in the QRDR of the gyrA gene and a single mutation in the parC gene. Active efflux pump mechanisms were also found to be involved in ciprofloxacin resistance. Finally, a large number of PFGE patterns (non-clonal genotypes) were observed among the S. Typhi isolates. In conclusion, a high rate of ciprofloxacin resistance was observed in comparison to other endemic areas in blood culture isolates of S. Typhi from Pondicherry, India, with steadily increasing NAR but decreasing MDR isolations over the study period. This is most likely to be due to an increased use of ciprofloxacin as a first-line drug of choice over more traditional antimicrobial agents for the treatment of typhoid fever.

Antimicrobial resistance in typhoidal salmonellae.

Infections with Salmonella are an important public health problem worldwide. On a global scale, it has been appraised that Salmonella is responsible for an estimated 3 billion human infections each year. The World Health Organization (WHO) has estimated that annually typhoid fever accounts for 21.7 million illnesses (217,000 deaths) and paratyphoid fever accounts for 5.4 million of these cases. Infants, children, and adolescents in south-central and South-eastern Asia experience the greatest burden of illness. In cases of enteric fever, including infections with S. Typhi and S. Paratyphi A and B, it is often necessary to commence treatment before the results of laboratory sensitivity tests are available. Hence, it is important to be aware of options and possible problems before beginning treatment. Ciprofloxacin has become the first-line drug of choice since the widespread emergence and spread of strains resistant to chloramphenicol, ampicillin, and trimethoprim. There is increase in the occurrence of strains resistant to ciprofloxacin. Reports of typhoidal salmonellae with increasing minimum inhibitory concentration (MIC) and resistance to newer quinolones raise the fear of potential treatment failures and necessitate the need for new, alternative antimicrobials. Extended-spectrum cephalosporins and azithromycin are the options available for the treatment of enteric fever. The emergence of broad spectrum ß-lactamases in typhoidal salmonellae constitutes a new challenge. Already there are rare reports of azithromycin resistance in typhoidal salmonellae leading to treatment failure. This review is based on published research from our centre and literature from elsewhere in the world. This brief review tries to summarize the history and recent trends in antimicrobial resistance in typhoidal salmonellae.

Molecular characterization of antimicrobial resistance in non-typhoidal salmonellae associated with systemic manifestations from India.

Extended-spectrum cephalosporins and fluoroquinolones are essential antimicrobials for treating invasive salmonellosis, although emerging resistance to these antimicrobials is of growing concern, especially in India. Therefore, a study was conducted to characterize the antimicrobial susceptibility phenotypes, types of extended-spectrum ß-lactamase (ESBL) gene plasmids and serological relationships of 21 non-typhoidal Salmonella isolates from patients who attended three different hospitals in India from 2006 to 2008. The isolates were cultured from stool, blood and cerebrospinal fluid samples obtained from patients presenting with diarrhoea and accompanying systemic manifestations such as fever, vomiting and meningism. Non-typhoidal Salmonella isolates were investigated using serotyping and antimicrobial susceptibility testing. PCR screening was also performed to detect the ß-lactamase, qnr and aac(6')-Ib-cr genes and class 1 integrons. Sequencing for quinolone resistance mutations and plasmid replicon typing were also performed. An antimicrobial resistance microarray was used for preliminary screening and identification of bla(TEM) and bla(SHV) genes, and phenotypic testing for the presence of efflux pumps was also performed. Ten out of 21 isolates (48%) possessed the extended-spectrum cephalosporin resistance phenotype, with PCR amplification and sequencing revealing that isolates possessed TEM-1, SHV-12, DHA-1, OXA-1-like and CTX-M-15 ESBL genes. FII(s) plasmid replicons were detected in seven isolates (33%). The involvement of efflux pumps was detected in four isolates (19%) resistant to ciprofloxacin. It was concluded that SHV-12-carrying Salmonella serotype Agona may play an important role in ESBL-mediated resistance in non-typhoidal salmonellae in India. The very high percentage (48%) of ESBL-producing non-typhoidal salmonellae isolated from these patients represents a real and immediate challenge to the effective antimicrobial therapy of Salmonella infections associated with systemic manifestations. Continued surveillance for the presence of ESBL-producing (non-typhoidal) salmonellae in India is essential.

Antimicrobial activity of highly stable silver nanoparticles embedded in agar-agar matrix as a thin film.

Highly stable silver nanoparticles (Ag NPs) in agar-agar (Ag/agar) as inorganic-organic hybrid were obtained as free-standing film by in situ reduction of silver nitrate by ethanol. The antimicrobial activity of Ag/agar film on Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), and Candida albicans (C. albicans) was evaluated in a nutrient broth and also in saline solution. In particular, films were repeatedly tested for antimicrobial activity after recycling. UV-vis absorption and TEM studies were carried out on films at different stages and morphological studies on microbes were carried out by SEM. Results showed spherical Ag NPs of size 15-25 nm, having sharp surface plasmon resonance (SPR) band. The antimicrobial activity of Ag/agar film was found to be in the order, C. albicans>E. coli>S. aureus, and antimicrobial activity against C. albicans was almost maintained even after the third cycle. Whereas, in case of E. coli and S. aureus there was a sharp decline in antimicrobial activity after the second cycle. Agglomeration of Ag NPs in Ag/agar film on exposure to microbes was observed by TEM studies. Cytotoxic experiments carried out on HeLa cells showed a threshold Ag NPs concentration of 60 µg/mL, much higher than the minimum inhibition concentration of Ag NPs (25.8 µg/mL) for E. coli. The mechanical strength of the film determined by nanoindentation technique showed almost retention of the strength even after repeated cycle.

Simple screening tests for detection of carbapenemases in clinical isolates of nonfermentative Gram-negative bacteria.

BACKGROUND & OBJECTIVE: The production of carbapenemases is an important mechanism responsible for the carbapenem resistance. A simple and inexpensive testing method for screening of carbapenemase producers is essential. A prospective study was undertaken to detect metallo-beta-lactamases (MBLs) and AmpC beta-lactamases in nonfermentative Gram negative bacteria and to evaluate the various methods for detection of carbapenemases and MBLs. METHODS: A total of 100 Acinetobacter spp. (78 A. baumannii and 22 A. lwoffii) and 140 Pseudomonas spp. (103 P. aeruginosa and 37 other Pseudomonas spp.) were screened for meropenem resistance by Kirby-Bauer disc diffusion method. Modified Hodge test, EDTA disk synergy (EDS) test and AmpC disk test were used for the detection of carbapenemases, MBLs and AmpC beta-lactamases, respectively. RESULTS: Forty six (59.0%) A. baumannii, 7 (31.8%) A. lwoffii, 32 (31.1%) P. aeruginosa and 7 (18.9%) Pseudomonas spp. were resistant to meropenem. Among the 32 meropenem resistant P. aeruginosa, 15 (46.9%) were AmpC beta-lactamase producers, 16 (50.0%) MBL producers by EDS test, but only 9 (28.1%) found positive for carbapenemases by modified Hodge test. Among the 46 meropenem resistant A. baumannii, 31 (67.4%) were AmpC beta-lactamase producers, 3 (6.5%) MBL producers, but only 1 (14.3%) was positive for carbapenemases by modified Hodge test. One P. aeruginosa was positive for carbapenemase by modified Hodge test, but was negative for MBL and AmpC beta-lactamase. INTERPRETATION & CONCLUSION: MBL production is an important mechanism of carbapenem resistance among Pseudomonas species but not among Acinetobacter species. EDS is more sensitive for detection of MBLs than modifi ed Hodge test. Both EDTA-meropenem and EDTA-ceftazidime combination must be used to detect all the MBL producers. Carbapenemases other than MBL may also be responsible for carbapenem resistance. AmpC beta-lactamase is also a contributory factor for carbapenem resistance among the isolates in the hospital.

Increased incidence of atypical Papanicolaou tests from ThinPreps of postmenopausal women receiving hormone replacement therapy.

BACKGROUND: The authors noticed a significant increase in the rate of atypical squamous cells of undetermined significance (ASCUS) diagnoses in postmenopausal women receiving hormone replacement therapy with ThinPrep (monolayer) Papanicolaou (Pap) tests (8.3%) as compared with conventional Pap tests (2.3%; P = 0.025). The purpose of this study was to correlate these morphologic changes with human papillomavirus (HPV) DNA detection by in situ hybridization and clinical outcome. METHODS: Twenty-five monolayer Pap tests diagnosed as ASCUS in postmenopausal women receiving hormone replacement, as well as 2 cases of low-grade squamous intraepithelial lesion (SIL) from the same group, were destained in acid alcohol. In situ HPV hybridization was performed with a consensus probe that can detect greater than 90% of the HPV types that occur in cervical SIL. In addition, 11 of the ASCUS tests were analyzed with the periodic acid-Schiff (PAS) stain. RESULTS: All 25 ASCUS tests were negative for HPV DNA as compared with a detection rate of 15 of 17(88%) for low-grade SIL. Glycogen that was PAS positive was present in the atypical squamous cells of the postmenopausal women for whom HPV was not detected. Clinical follow-up demonstrated that only 2 of the 25 women with ASCUS had a subsequent biopsy proven SIL. CONCLUSIONS: The ThinPrep preparation can produce artifactual HPV-like changes in postmenopausal women receiving hormone replacement therapy that may be caused by glycogenization and, because of the high incidence of HPV negativity and negative clinical outcome, are best considered as benign cellular changes.

Chronic ethanol consumption impairs spatial remote memory in rats but does not affect cortical cholinergic parameters.

We have studied learning, memory and cortical cholinergic parameters after oral administration of 20% v/v ethanol solution to male Fisher rats for 6 months. A group of rats were trained to behave efficiently in an eight-arm radial maze and after that split into two subgroups submitted to ethanol or control treatment. Ethanol-treated rats had more difficulty in relearning the same task 1 year later, compared to ethanol-untreated rats (control). Differences in working memory performance were found, but only in the first 10 training sessions. Another group of rats, which had not been pretrained, was also split into two subgroups submitted to ethanol or control treatment. After that, these rats were trained in the radial maze task for the first time. No significant difference was found between the reference memory performance of the untreated subgroup and the treated one. These two subgroups did not significantly differ in their working memory performance either. Moreover, there were no significant differences between treated and control subjects in the following biochemical brain cortical parameters: in vitro acetylcholinesterase (AChE) activity, and stimulated acetylcholine (ACh) release. This work presents an experimental design that allows assessment of remote memory performance after ethanol chronic consumption and shows that the experimental subject is able to retain the behaviors learned 1 year before. It was concluded that chronic ethanol treatment may cause retrograde amnesia, which does not seem to be linked with a cortical cholinergic deficit.