Bacteriophages as therapeutic & disinfectant agents to tackle multidrug-resistant Acinetobacter baumannii

Background & objectives: Multidrug-resistant (MDR) Acinetobacter baumannii is a serious threat for human health worldwide. The studies on agents targeting A. baumannii are imperative due to identified A. baumannii co-infections in COVID-19. Bacteriophages are promising antibacterial agents against drug-resistant bacteria. This study intended to isolate bacteriophages against MDR A. baumannii from the water of river Ganga, to be used potentially as therapeutic and disinfectant particles. Methods: Acinetobacter phages were isolated from the Ganga water collected from Kanpur and further tested on 50 MDR A. baumannii isolates to determine host range. The phages were morphologically characterized by transmission electron microscopy. The disinfectant property of the isolated phages was tested by spraying of bacteriophage cocktail on MDR A. baumannii contaminated plastic surface, analyzed by colony-forming unit (CFU) and bioluminescence assay (adenosine triphosphate monitoring). Results: A total of seven bacteriophages were isolated against MDR A. baumannii. The bacteriophages lysed three MDR A. baumannii isolates out of 50 tested, showing narrow host range. Electron microscopy revealed hexagonal heads and long tails of bacteriophages, belonging to order Caudovirales. The bacteriophage cocktail reduced the MDR A. baumannii load efficiently on plastic surface, evidenced by reduction in CFUs and bioluminescence. Interpretation & conclusions: The findings of this study suggest that the isolated bacteriophages are potential lytic agents for MDR A. baumannii clinical isolates, and may be used as potential therapeutic agents as well as disinfectant to combat MDR A. baumannii with due consideration to phage host specificity, with further characterization.n

Exploring the Clinical Variables among Drug-Dependent HIV-Positive Patients: Implications for Treatment Strategies

This study explored the profile of HIV positive patients seeking treatment at a tertiary care addiction treatment facility. A retrospective study was done to collet detailed information on clinical characteristics: drug use (type, age of initiation, duration), general medical condition and past treatment history. The study included 138 patients with mean (SD) age 30.2 (8.3) years. Opioid dependence with injecting drug use (IDU) was diagnosed in 97% of the patients. The median age of injecting onset was 24.5 years (IQR 20-31 years). The most frequently injected substances were pheniramine (60.1%) and buprenorphine (59.4%). Past treatment seeking was reported by 57% patients and interestingly they were less likely to present any medical condition (2 =69.611, p < 0.001). Variability in the age of onset of drug use indicates the need for broad based approach to prevent IDU and motivation to seek treatment may lead to better health conditions.

The Clinical Profile and Risk Factors of Pneumonia in Children

Background: The problem of increased ARI morbidity and mortality has arisen as a result of modernization, industry, and urbanization. There is a deficiency of epidemiological studies on risk factors and treatment. There is a significant vacuum in our understanding of these issues, which must be filled by methodical research. The purpose of this study is to evaluate the risk factors developing pneumonia in our area.Material & Methods:This was a prospective clinical study of pneumonia conducted on 90 children who were admitted to Paediatric ward in Abdul MalekUkil Medical College, Noakhali, Bangladesh in study duration. Epidemiological factors affecting the same were studied and bronchoscopy was done whenever it was needed. A detailed history of the relevant symptoms, such as fever, cough, rapid breathing, refusal of feeds, noisy breathing, bluish discolouration etc., was collected.Results:The most affected children belonged to the age group of 1 year to 3 years (64.9%). Bronchopneumonia (86.2%) was the most common clinical diagnosis made at admission. According to WHO ARI control programme, 28.7% had pneumonia, 54.3% had severe pneumonia and 17% very severe pneumonia. It was found that younger age group, malnutrition, kutcha house, crowding, poor sanitation facilities, cooking with fuel other than LPG (indoor pollution) and low socio economic status and high respiratory rate were significant risk factors for pneumonia in children.Conclusions:One of the leading causes of mortality and death in children is particularly pneumonia. In newborns and preschool children, bronchopneumonia is the most common symptom.

Risk Factors for Central line – associated Bloodstream Infections.

Objective: To carry out surveillance of central line – associated bloodstream infections in a Pediatric intensive care unit (PICU) and determine associated risk factors. Methods: This prospective study was conducted over 1.5 years in the PICU. CDC definitions for these infections were followed and associated risk factors were identified. Results: Of 265 enrolled children with central line, 13 developed blood stream infections (incidence density 5.03/1000 central-line days). Significant risk factors included changing the central-line, especially triple lumen, and frequently accessing the central-line. Conclusion: Central-line associated bloodstream infections are preventable primary bacteremias and intervention strategies for prevention should be based on evidence generated to devise future protocols.

Hand Hygiene Compliance of Healthcare Workers in a Pediatric Intensive Care Unit.

We conducted an observational study over 11 months to assess the hand-hygiene compliance of health-care workers in a Pediatric intensive care unit. The overall compliance was 80.9%, which decreased with increase in workload (79.2% vs. 82.9%). Assessment of hand hygiene compliance helps understand the gaps in practices followed by healthcare workers, and plan effective protocols.

Induction of resistant mutants of Salmonella enterica serotype Typhi under ciprofloxacin selective pressure.

Background & objectives: Infection with Salmonella enterica serovar Typhi (hereafter S. Typhi) is an important public health problem in India. There has been an increase in the number of reported clinical failures to ciprofloxacin treatment but the data on possible mechanism of failure are limited. One mechanism that has been widely reported and found associated with ciprofloxacin resistance, is the mutations in target genes in QRDR (quinolone resistance determining region). It is hypothesized that mutations in DNA gyrase or topoisomerase IV result in therapeutic failure under selective pressure of antibiotic while the patient is on treatment. We undertook in vitro sequential selection studies to expose the clinical isolates of S. Typhi to different concentration of ciprofloxacin to study the role of antibiotic selective pressure in the development of mutations in QRDR. Methods: Total 26 clinical isolates were divided in to two parts: part I included six isolates obtained from three patients with relapse of enteric fever and part II included 20 isolates with different ciprofloxacin MIC levels. For in vitro induction of mutation experiment, five S. Typhi isolates were selected which included three NAS (nalidixic acid sensitive) and 2 NAR (nalidixic acid resistant) S. Typhi. These isolates were grown under increasing concentrations of ciprofloxacin and mutations acquired in QRDR of DNA gyrase (gyrA and gyrB) and topoisomerase IV (parC and parE) were investigated by sequencing. Results: For the isolates included in the part I of the study, it was found that the MIC to ciprofloxacin increased in the isolates obtained during the relapse of enteric fever as compare to the first isolate. All isolates had single mutation in gyrA gene at S83 without additional mutation in the second isolate. In the second part of the study, the nine isolates with varying MICs to ciprofloxacin also had single mutation in gyrA gene at S83 and another six had triple mutations, two mutations in gyrA gene (at S83 and D87) and one mutation in parC gene (at S80). In in vitro induction of mutation experiment, all mutated isolates showed triple mutation (two mutation in gyrA and one in parC gene) while no mutations were found in wild isolates. Interpretation & conclusions: Upon exposure to the step-wise increased concentration of ciprofloxacin, isolates become more tolerant to the ciprofloxacin and showed 2-4 fold higher MICs without new mutation after 8 μg/ml. So the accumulation of mutations under continuous ciprofloxacin pressure and tolerance of the mutant isolates led to the clinical failure. These results also suggested that there could be another mechanism responsible for resistance.

Prospective study estimating healthcare associated infections in a paediatric hemato-oncology unit of a tertiary care hospital in north India.

Background & objectives: Healthcare associated infections (HAIs) are responsible for morbidity and mortality among immunocompromised and critically ill patients. We undertook this study to estimate the burden of HAIs in the paediatric cancer patients in a tertiary care hospital in north India. Methods: This prospective, observational study, based on active surveillance for a period of 11 months was undertaken in a 4-bedded isolated, cubicle for paediatric cancer patients. Patients who stayed in the cubicle for ≥48 h, were followed prospectively for the development of HAIs. Results: Of the 138 patients, 13 developed 14 episodes of HAIs during the study period. Patient-days calculated were 1273 days. Crude infection rate (CIR) and incidence density (ID) of all HAIs were 9.4/100 patients and 11/1000 patient-days, respectively. Of the 14 episodes of HAIs, seven (50%) were of blood stream infections (HA-BSI), five (36%) of pneumonia (HAP) and two (14%) urinary tract infections (HA-UTI). The CIRs of HA-BSI, HAP and HA-UTI were 5.1, 3.6 and 1.4/100 patients, respectively. The corresponding IDs were 5.5, 3.9 and 1.6/1000 patient-days, respectively. Mean length of stay was significantly higher in patients who developed an HAI [13.8 (range 7-30), median (Interquartile range) 12 (11-14)] vs 7.5 days [range 2-28, median (interquartile range) 7 (5-9); P<0.0001]. Also mortality was significantly higher in patients who developed an HAI [23% (3/13) vs 3% (4/125), P<0.05]. Interpretation & conclusions: The incidence of HAIs in the paediatric cancer patients in the study was 11/1000 patient days, of which HA-BSIs were the commonest. HAIs were associated with an increase in morbidity and mortality amongst this high risk patient population.

Multiple locus sequence typing of Salmonella Typhi, isolated in north India - a preliminary study.

Background & objectives: In India enteric fever is a major public health problem and Salmonella Typhi is the most common aetiologic agent. Any control strategy for such infections depends to a large extent on the understanding of the disease and relatedness of strains across the world. Multi locus sequence typing (MLST) is one such method of genotyping of bacteria based upon housekeeping genes of known function and chromosome position. MLST data of pathogens are important to determine the molecular evolution by a stable and reproducible method. This study was undertaken to determine the sequence types of representatives S. Typhi isolates obtained from enteric fever patients in a tertiary care centre in north India, over a period of 20 years (1990-2010). Methods: A total of 30 representative isolates of S. Typhi identified by biochemical and serological tests were subjected to multi locus sequence typing (MLST). Seven housekeeping genes of known function and chromosome position were used for the typing by MLST. Sequencing was carried out by using an automated DNA sequencer and results were analyzed to generate phylogenetic tree. Results: MLST pattern grouped S. Typhi into two sequence types- ST1 and ST2. ST1 was predominantly present followed by ST2. Interpretation & conclusions: By MLST the presence of both sequence types, ST1 and ST2, was found in S. Typhi isolates in our region. Predominately ST1 was present followed by ST2. These preliminary results corroborate the global distribution of both sequence types of S. Typhi and also emphasize for the continuous screening of S. Typhi.

Characterization of nasopharyngeal isolates of type b Haemophilus influenzae from Delhi.

Background & objectives: Haemophilus influenzae is an important cause of mortality and morbidity among young children in developing countries. Increasing incidence of antibiotic resistance especially production of extended spectrum beta lactamase (ESBL) has made treatment and management of H. influenzae infection more difficult. Nasopharyngeal H. influenzae isolates are excellent surrogate for determination of antibiotic resistance prevalent among invasive H. influenzae isolates. In this study, we characterized nasopharyngeal H. influenzae isolates obtained from healthy school going children in Delhi. Methods: Nasopharyngeal H. influenzae isolates were collected from healthy school going children and subjected to serotyping, fimbrial typing and antibiogram profiling. ESBL production was recorded using phenotypic as well as molecular methods. Multi locus sequence typing (MLST) of 13 representative nasopharyngeal H. influenzae isolates was performed as per guidelines. Results: A significant proportion (26 of 80, 32.5%) of nasopharyngeal isolates of H. influenzae were identified as serotype b. Fimbrial gene (hifA) was detected in 23 (28.75%) isolates. Resistance against commonly prescribed antibiotics (Amp, Tet, Chloro, Septran, Cephalexin) were observed to be high among the nasopharyngeal commensal H. influenzae. Extended spectrum beta lactamase (ESBL) production was observed in a five (6.25%) isolates by both double disk diffusion and molecular typing. MLST identified several novel alleles as well as novel sequence types. Interpretation & conclusions: Our findings showed high resistance against common antibiotics and detection of ESBL in nasopharyngeal H. influenzae isolates collected from normal healthy school going children in Delhi. Detection of H. influenzae type b capsular gene and the presence of fimbrial gene (hif A) suggest virulence potential of these isolates. Discovery of novel alleles and presence of new sequence types (STs) among nasopharyngeal H. influenzae isolates may suggest wider genetic diversity.

Primary meningococcal polyarthritis in a young man.

We report a rare occurrence of primary meningococcal polyarthritis in a 19-year-old man. The fluid in the elbow joint showed Gram-negative diplococci but the culture was sterile. The diagnosis was confirmed by polymerase chain reaction targeting crgA gene of Neisseria meningitidis.

Prevalence of cholera in pediatric patients with acute dehydrating diarrhea.

Objective. To estimate the prevalence of culture-confirmed cholera in patients with acute dehydrating diarrhea, at a tertiary care center in north India, during a 6-month period from March to August, 2006. Methods. We studied 145 children, who presented to the pediatric emergency services of a tertiary care teaching hospital in north India with acute dehydrating diarrhea. Each patient had his/her stool sample collected for Vibrio cholerae culture and hanging drop preparation for darting motility. The stool specimen for hanging drop analysis was immediately transported to the emergency laboratory, where a trained technician prepared the slides and examined them for darting motility characteristic of Vibrio cholerae. Results. V. cholerae was isolated in 36 (24.8%) patients. Forty-nine (33.7%) patients had a positive hanging drop examination. Hanging drop examination had a sensitivity and specificity of 85.8% and 81.7%, respectively. Severe dehydration (OR 4.3; P<0.01) and hanging drop positivity (OR 12.42; P<0.001) were associated with higher odds of cholera after adjustment for other risk factors. Conclusion. Cholera is an important cause of acute watery diarrhea in pediatric patients in urban north India and should be ruled out in all children presenting with acute dehydrating diarrhea, particularly those with severe dehydration. Hanging drop test is useful for diagnosis in the emergency setting.

Occurrence of TEM & SHV gene in extended spectrum beta-lactamases (ESBLs) producing Klebsiella sp. isolated from a tertiary care hospital.

BACKGROUND & OBJECTIVE: Extended-spectrum beta-lactamases (ESBLs) are rapidly evolving group of beta-lactamase enzymes produced by the Gram negative bacteria. These enzymes have been derived from TEM and SHV genes by mutations and have been well described in Klebsiella pneumoniae. Information on molecular types of ESBL positive Klebsiella sp. is lacking from India. We therefore undertook this study to look for the TEM and SHV genes in ESBL positive Klebsiella sp. isolated from the patients admitted to a tertiary care hospital in north India. METHODS: A total of 204 multidrug-resistant isolates of Klebsiellae obtained from clinical samples; blood (n=108), urine (n=15), pus (n=2) and sputum (n=79) were obtained and screened for resistance to 3rd generation cephalosporins (3GC). The ESBL status was determined by double disk diffusion test (DDDT) and further by ESBL E-test. Multiplex PCR specific for TEM and SHV genes was performed to distinguish four different genotypes: TEM-positive, SHV-positive, TEM- and SHV-positive and non-TEM non-SHV ESBL types. RESULTS: Eighty six per cent (175 of 204) of the isolates were found to be resistant to at least one of the 3GCs, of which 97.1 per cent (170) of Klebsiella sp. isolates were confirmed to be positive for ESBL. Of these 170 isolates, 95 were randomly selected for PCR of TEM and SHV genes. Isolates having both TEM and SHV genes were common (67.3%) whereas only 20 per cent isolates possessed TEM gene and 8.4 per cent SHV gene alone. INTERPRETATION & CONCLUSION: Our findings showed that the majority of the ESBL positive clinical isolates of Klebsiella sp. carried both TEM and SHV genes followed by TEM alone. Such studies need to be done in various geographical regions of the country to know about the prevalent genotypes for better management of infection.

Profile of bacterial isolates from suspected cases of infective endocarditis (IE): a retrospective study in a north Indian tertiary care hospital.

Infective Endocarditis (IE) is an emerging infection of the twenty-first century. This chronic Infection is mainly caused by bacteria, although fungi can also be associated with it. It is Important to know the profile of bacteria causing IE in a given region so as to suggest the empirical therapy for this serious illness. Blood culture isolates of clinically diagnosed or suspected cases of IE admitted to various wards of the All India Institute of Medical Sciences were analyzed retrospectively from January 2000 to June 2004. Standard techniques were used for the isolation and identification of the bacteria. Our study has demonstrated the predominance of Gram-negative bacilli, especially, Acinetobacter species and Pseudomonas aeruginosa, which are notorious for antimicrobial resistance, as the aetiological agents of IE. Amongst Gram-positive cocci, Enterococci exhibiting HLAR comprised the predominant species. Methicillin resistance among staphylococcal strains in this Tertiary care hospital is adding to the therapeutic challenge in the management of this serious illness. Although antimicrobial treatment should not be delayed in such cases, we cannot undermine the importance of isolation and identification of the etiological agents and the determination of the antimicrobial susceptibility for the management of these life-threatening conditions as well as for the formulation of guidelines for empirical therapy of these cases.

Emerging resistance to carbapenems in a tertiary care hospital in north India.

BACKGROUND AND OBJECTIVES: Carbapenems are beta-lactam antibiotics, presently considered as most potent agents for treating multi-drug resistant Gram-negative bacilli infections. In India carbapenems available for use are meropenem and imipenem, introduced recently. Resistance to these has been reported in a few bacteria especially Pseudomonas spp. We therefore retrospectively evaluated the antibiotic susceptibility pattern to these agents amongst various clinical isolates in a tertiary care hospital in north India. METHODS: In this study Gram-negative bacterial pathogens isolated from clinical samples were tested for extended spectrum beta lactamase (ESBL) production. All ESBL positive bacteria were tested for meropenem and imipenem activity pattern using NCCLS guidelines. A total of 2626 consecutively isolated Gram-negative bacteria, which tested positive for ESBL production by the double disk diffusion method, were included. RESULTS: The different bacteria isolated were Pseudomonas spp. 759, Acinetobacter spp. 676, Escherichia coli 569, Klebsiella spp. 343, Enterobacter spp. 150, Citrobacter spp. 57 and Proteus spp. 72. Overall resistance to meropenem was more (22.16%) than imipenem (17.32%). Maximum resistance was seen in Pseudomonas spp. M(R) 37.6 per cent, I(R) 30 per cent. In isolates from intensive care units (ICU) resistance to carbapenems was significantly higher than non-ICU patients. INTERPRETATION AND CONCLUSION: Resistance to meropenem and imipenem was seen in various clinical isolates of Gram-negative ESBL-positive bacteria. There is a need to alarm our clinicians for judicious use of antibiotics.