Utility of lytic bacteriophage in the treatment of multidrug-resistant Pseudomonas aeruginosa septicemia in mice.

Drug resistance is the major cause of increase in morbidity and mortality in neonates. One thousand six hundred forty-seven suspected septicemic neonates were subjected for microbiological analysis over a period of 5 years. Forty-two P. aeruginosa were isolated and the antibiogram revealed that 28 P. aeruginosa were resistant to almost all the common drugs used (multidrug-resistant). The emergence of antibiotic-resistant bacterial strains is one of the most critical problems of modern medicine. As a result, a novel and most effective approaches for treating infection caused by multidrug-resistant bacteria are urgently required. In this context, one intriguing approach is to use bacteriophages (viruses that kill bacteria) in the treatment of infection caused by drug-resistant bacteria. In the present study, the utility of lytic bacteriophages to rescue septicemic mice with multidrug-resistant (MDR) P. aeruginosa infection was evaluated. MDR P. aeruginosa was used to induce septicemia in mice by intraperitoneal (i.p.) injection of 10(7) CFU. The resulting bacteremia was fatal within 48 hrs. The phage strain used in this study had lytic activity against a wide range of clinical isolates of MDR P. aeruginosa. A single i.p. injection of 3 x 10(9) PFU of the phage strain, administered 45 min after the bacterial challenge, was sufficient to rescue 100% of the animals. Even when treatment was delayed to the point where all animals were moribund, approximately 50% of them were rescued by a single injection of this phage preparation. The ability of this phage to rescue septicemic mice was demonstrated to be due to the functional capabilities of the phage and not to a nonspecific immune effect. The rescue of septicemic mice could be affected only by phage strains able to grow in vitro on the bacterial host used to infect the animals and when such strains are heat-inactivated, they lose their ability to rescue the infected mice. Multidrug-resistant bacteria have opened a second window for phage therapy. It would seem timely to begin to look afresh at this approach. A scientific methodology can make phage therapy as a stand-alone therapy for infections that are fully resistant to antibiotics.

Bacterial meningitis: rapid diagnosis and microbial profile: a multicentered study.

Meningitis continues to be a formidable illness with high morbidity and mortality among children in India. The present study was undertaken to prospectively look for the prevalence of pyogenic meningitis at Gulbarga and to find out the utility of gram stain, Latex Agglutination Test and (LTA) and C-reactive protein in the rapid diagnosis of pyogenic meningitis from children. Over a 48-months period, 535 children with a presumptive clinical diagnosis of acute bacterial meningitis were investigated by direct microscopy, CRP, bacterial culture, latex agglutination test (L TA), cell count and cell type and biochemical tests. Latex Agglutination Test (LA T) was done for detection of the antigens of Streptococcus pneumoniae, Group B Streptococci, E. coli, Neisseria meningitidis and Haemophilus influenzae type b. Among 535 suspected meningitis cases, 291 cases were diagnosed as pyogenic meningitis cases based on biochemical tests, cell count and cell type. Out of 291 cases, 55 cases have already received antibiotic treatment. Among 236 cases of untreated pyogenic meningitis cases, 199 CSF samples were culture positive. Streptococcus pneumoniae (44.7%) was the predominant organism identified, followed by H influenzae (25.6%) and Gp. B. Streptococci (9.5%). 208 of 236 cases were gram-stain positive, 129 cases had elevated CSF-CRP and 214 cases were diagnosed as pyogenic meningitis by the detection of bacterial antigens by latex agglutination test. Among 55 pretreated cases, only 05 (9.1%) CSF samples were culture positive, bacteria was observed in 36 gram stain smear, CRP was elevated in 16 CSF samples and 52 pretreated cases of suspected meningitis were diagnosed as pyogenic meningitis by latex agglutination test for detection of bacterial antigens. Many of the bacterial isolates were sensitive to gentamicin, cefotaxime and ceftriaxone and least sensitive to tetracycline and gentamicin. 13.1% of gram-negative bacilli were ESBL producers. To conclude, inclusion of latex agglutination test for detection of bacterial antigen in the routine diagnosis adds a valuable adjunct in the rapid and accurate diagnosis of pyogenic meningitis.

Emergence of extended spectrum beta lactamase mediated resistance in neonatal septicemia.

This study was carried out in microbiology department of Gulbarga university, Gulbarga to find out the frequency of extended spectrum beta lactamase (ESbL) producing gram-negative bacilli isolated from neonatal septicemic cases. This study was carried out from Oct 2001 to June 2004. A total of 471 consecutive Gram-negative bacilli were recovered during the study period from blood samples. Extended spectrum beta lactamase detection in gram-negative isolates was carried out by double disk synergistic method on Mueller Hinton agar. A susceptibility disk containing amoxicillin-clavulanate was placed as the inhibitor of beta lactamase in the center of the plate, and cefotaxime, ceftazidime, ceftriaxone and aztreonam disks were placed 30 mm (center to center) from the amoxicillin-clavulanate disk. Enhancement of the zone of inhibition of the oxyimino-lactam caused by the synergy of the clavulanate in the amoxicillin-clavulanate disk was considered as evidence of ESbL production. Escherichia coli ATCC 25922 and K. pneumoniae ATCC 700603 were used as control strains. The frequency of ESbL producing gram-negative bacilli among the neonatal septicemic cases was 22.7%.

Bacteriophage in the treatment of experimental septicemic mice from a clinical isolate of multidrug resistant Klebsiella pneumoniae.

Drug resistance is the major cause of increase in morbidity and mortality in neonates. The emergence of antibiotic-resistant bacterial strains requires the exploration of alternative antibacterial therapies and the concern that human kind in re-entering the 'pre-antibiotic era' has become very real and the development of alternative anti-infection modalities has become one of the highest priorities of modern medicine and biotechnology. This has spurred biomedical researchers to expand their efforts to identify new technologies and products that employ novel mechanism of action against the "super-bugs". One of such alternatives stems up from an old idea is the bacteriophage therapy, which led our group to study the ability of bacterial viruses (bacteriophages or phages) to rescue septicemic mice with multidrug resistant (MDR) Klebsiella pneumoniae isolated from neonatal septicemia. The phage strain used in this study had lytic activity against a wide range of clinical isolates of MDR Klebsiella pneumoniae. One of these MDR Klebsiella strain was used to induce septicemia in mice by intraperitoneal (i.p.) injection of 10(9) CFU. The resulting bacteremia was fatal within 48 h. A single i.p. injection of 3x10(8) PFU of the phage strain administered 45 min after the bacterial challenge, was sufficient to rescue 100% of the animals. Even when treatment was delayed to the point where all animals were moribund, approximately 50% of them were rescued by a single injection of this phage preparation. The ability of this phage to rescue septicemic mice was demonstrated to be due to the functional capabilities of the phage and not to a nonspecific immune effect. The rescue of septicemic mice could be affected only by phage strains able to grow in vitro on the bacterial host used to infect the animals and when such strains are heat inactivated they lose their ability to rescue the infected mice.

Incidence and antifungal susceptibility of Candida species in neonatal septicemia.

Septicemia is the leading cause of morbidity and mortality in neonates. In this study, 828 consecutive neonates suspected of having septicemia from various neonatal intensive care unit hospitals in Gulbarga, Karnataka were investigated for isolation of microorganisms. Two samples of blood were collected aseptically for isolating the etiology. The cultures were positive in 346 (41.9%) cases for aerobic bacteria and 68 (8.3%) for Candida species. Among Candida species, C. tropicalis--27(39.7%) was the predominant organism followed by C. albicans 16(23.5%) and C. guillermondi 13(19.1%). The standard macrobroth dilution method was carried out to determine the minimum inhibitory concentration (MIC); C. krusei ATCC 6258 standard strain was included for quality control purpose. 4(25.0%) stains of C. albicans were resistant to amphotericin-beta and 2(12.5%) to fluconazole. High-level resistance to fluconazole was observed in C. krusei 4(40.0%).