Lipika Singhal

Burkholderia cepacia complex: Beyond pseudomonas and acinetobacter

Burkholderia cepacia complex (BCC) is an important nosocomial pathogen in hospitalised patients, particularly those with prior broad-spectrum antibacterial therapy. BCC causes infections that include bacteraemia, urinary tract infection, septic arthritis, peritonitis and respiratory tract infection. Due to high intrinsic resistance and being one of the most antimicrobial-resistant organisms encountered in the clinical laboratory, these infections can prove very difficult to treat and, in some cases, result in death. Patients with cystic fibrosis (CF) and those with chronic granulomatous disease are predisposed to infection by BCC bacteria. BCC survives and multiplies in aqueous hospital environments, including disinfectant agents and intravenous fluids, where it may persist for long periods. Outbreaks and pseudo-outbreaks of BCC septicaemia have been documented in intensive care units, oncology units and renal failure patients. BCC is phenotypically unremarkable, and the complex exhibits an extensive diversity of genotypes. BCC is of increasing importance for agriculture and bioremediation because of their antinematodal and antifungal properties as well as their capability to degrade a wide range of toxic compounds. It has always been a tedious task for a routine microbiological laboratory to identify the nonfermenting gram-negative bacilli, and poor laboratory proficiency in identification of this nonfermenter worldwide still prevails. In India, there are no precise reports of the prevalence of BCC infection, and in most cases, these bacteria have been ambiguously reported as nonfermenting gram-negative bacilli or simply Pseudomonas spp. The International Burkholderia cepacia Working Group is open to clinicians and scientists interested in advancing knowledge of BCC infection/colonisation in persons with CF through the collegial exchange of information and promotion of coordinated approaches to research.

Trends in antimicrobial susceptibility of Salmonella Typhi from North India (2001-2012)

PURPOSE Enteric fever is endemic in India with Salmonella Typhi being the major causative agent. Antibiotic therapy constitutes the mainstay of management. The present study was undertaken to find the susceptibility profile of Salmonella enterica var Typhi (S. Typhi) blood isolates in a tertiary care hospital between January 2001 and December 2012. MATERIALS AND METHODS A retrospective analysis of laboratory records was carried out. Conventional blood culture method was used until 2009; from January 2010 onwards BACTEC 9240 system has been in use. Salmonella were confirmed by serotyping using group and type specific antisera. Antibiotic susceptibility was performed using the disk diffusion method. In addition 116 isolates were subjected to minimum inhibitory concentration testing for chloramphenicol, ciprofloxacin, amoxicillin and nalidixic acid (NA) using agar dilution and for ceftriaxone and azithromycin using E-strips (Biomerieux). RESULT A total of 1016 typhoidal salmonellae were obtained. The predominant serotype obtained was S. Typhi (852, 83.8%) followed by Salmonella enterica var Paratyphi A (164, 16.2%). We observed a re-emergence of susceptibility to first line antibiotics and a notable decline in multidrug resistant (MDR) strains. We also found all recent isolates resistant to NA and susceptible to third generation cephalosporins and 84.5% of isolates having decreasing ciprofloxacin susceptibility using revised criteria as per Clinical and Laboratory Standards Institute 2012 guidelines. CONCLUSION There has been re-emergence of susceptibility to first line antibiotics and a notable decline in MDR strains of S. Typhi. We have a very high resistance to NA and decreasing susceptibility to ciprofloxacin. Third generation cephalosporins and azithromycin seem to be effective therapeutic options. Judicious use of these antibiotics is mandatory to prevent emergence of resistant strains.

Antimicrobial activity of cefepime-tazobactam combination tested against clinical isolates of Enterobacteriaceae.

Antimicrobial activity of cefepime–tazobactam combination tested against clinical isolates of Enterobacteriaceae

Parvovirus B19 infection in pediatric patients with hematological disorders

Sir, Parvovirus B19 (PVB19) is a childhood infectious disease of global proportions. It has specificity for red blood cell precursors and acute infection causes cessation of erythropoiesis for 5-7 days. Apoptosis of red cell precursors in the bone marrow decreases the hemoglobin level for 1-2 weeks until the bone marrow recovers.[1] Children with hematological malignancies receiving chemotherapy and children with chronic hemolytic disorders are at a higher risk of acquiring PVB19 infection.[1] We aimed to study the frequency of acute PVB19 infection and its implications in 50 children (age ≤ 15 years) with various hematological disorders in the period between January 2009 and July 2011. These included: aplastic anemia/pancytopenia 31), thalassemia (2), immune thrombocytopenia (ITP) (8), Henoch-Schönlein purpura (HSP) (2), leukemia (6), and Hodgkins lymphoma (1). Fifty febrile children with no hematological disorders were included as a control group. These children were assessed clinically and the serum samples were collected and tested for presence of anti-PVB19-specific IgM antibody by ELISA (NovaTec Immunodiagnostics, Germany). Recent PVB19 infection, as determined by the presence of serum IgM antibodies, was found in 20 percent (10/50) of these children with hematological disorders compared to none in the control group. All PVB19-infected children presented with fever and a sudden unexplained worsening of anemia. PVB19 infection was considered when the children had a sudden increase in need of blood transfusions of up to once a week during the non-intensive phase of chemotherapy in the absence of any obvious infection/blood loss. There was no other symptom. No child with PVB19 died. All patients showed marked improvement with increase in hemoglobin over a period of 3-6 weeks. Management was supportive in the form of blood transfusions. One child was given Intra Venous Immunoglobulin (IVIg). It is known that individuals with decreased erythrocytosis seen in chronic hemolytic disorders, such as thalassemia, autoimmune hemolytic anemia, and in children with hematological malignancies who are anemic due to malignant infiltration of bone marrow and cytotoxic drugs, can develop transient aplastic crisis if infected with PVB19.[2-4] This virus has specificity for red blood cell precursors and can cause a complete cessation of erythrocyte production. This can aggravate anemia, worsen the general condition of patients, and in severe cases can lead to life-threatening complications such as circulatory collapse, congestive heart failure, cerebrovascular accident, or acute splenic sequestration[1] Serum PVB19-DNA and specific IgM can be determined for accurate diagnosis. However, since DNA may circulate for a few days (7-12 days) only, detection of IgM antibodies is a more reliable indicator of recent PVB19 infection.[5]

Reliability of Kirby-Bauer Disk Diffusion Method for Detecting Carbapenem Resistance in Acinetobacter baumannii-calcoaceticus Complex Isolates

We recently read an article on carbapenem susceptibility testing errors in studies that used three automated systems, disk diffusion (DD), Etest (ET), and broth microdilution (BMD), to investigate Acinetobacter baumannii-calcoaceticus complex (ABC) isolates ([1][1]). The authors concluded from the

In Vitro Activity of Doripenem against Burkholderia cepacia Complex Isolates from Non-Cystic Fibrosis Patients

Burkholderia cepacia complex (BCC), a group of nonfermenting Gram-negative bacilli (NFGNB), is emerging as an important nosocomial pathogen. It causes a wide variety of infections, such as pneumonia (especially in patients with cystic fibrosis), meningitis, and septicemia. These infections are often difficult to treat due to high intrinsic antimicrobial resistance in BCC and the lack of effective antibiotics. BCC is resistant to aminoglycosides, antipseudomonal penicillins, and cephalosporins (4). Hence, the need for new agents active against this pathogen has become critical for patient care. In recent years, carbapenems have assumed a greater therapeutic role for multidrug-resistant (MDR) isolates. Doripenem is a new broad-spectrum intravenous 1-β-methyl carbapenem. It has high activity against many Gram-positive and -negative organisms, including NFGNB. Against Pseudomonas aeruginosa, doripenem exhibits rapid bactericidal activity, with 2- to 4-fold-lower MICs than those of meropenem. However, the activity of doripenem against BCC has not been reported much, and present susceptibility reports are mainly from isolates of cystic fibrosis patients (6). Isolation of BCC from blood of non-CF septicemic patients is on the rise in our region (3), and the in vitro activity of doripenem against these isolates has been studied. Fifty blood isolates of BCC obtained between May 2007 and January 2010 were included in this study. These isolates were identified using standard biochemical tests and confirmed using recA PCR-based restriction fragment length polymorphism (RFLP) analysis as described by Mahenthiralingam et al. to identify species (5). B. cenocepacia (genomovar IIIA) was the most prevalent species of BCC obtained. Pure cultures of the isolates were lyophilized and stored at 4°C. The meropenem susceptibilities of these 50 isolates were determined by the disk diffusion method (Astra Zeneca Pvt. Ltd., India) according to CLSI guidelines, with 8 isolates (16%) being meropenem resistant. In vitro susceptibility to doripenem (Ranbaxy Intensiva, India) was determined by the Etest. Etest strips (bioMerieux India Pvt. Ltd.) were used per the instructions of the manufacturer. In addition, Escherichia coli ATCC 25922 and P. aeruginosa ATCC 27853 were tested for quality control, and MICs obtained for these organisms were within the ranges recommended by the CLSI (2a). A distribution of the MICs of doripenem determined by the Etest for 50 strains of BCC is shown in Fig. ​Fig.11. FIG. 1. Distribution of the MICs of doripenem determined by Etest for 50 strains of BCC. Black bars represent meropenem-resistant isolates. The MIC values classifying isolates as susceptible or resistant to doripenem suggested by Bhavnani et al. (2) and Andes et al. (1) indicate susceptibility at ≤4 mg/liter and resistance at ≥16 mg/liter, with intermediate resistance being inferred as 8 mg/liter. Since there are no FDA- or CLSI-approved breakpoints at this time for doripenem, the MIC values obtained were interpreted based on the above criteria, with all 50 isolates (100%) of BCC being susceptible to doripenem (MIC ≤ 4 mg/liter). A greater proportion of strains among meropenem-resistant BCC were inhibited by doripenem at 2 mg/liter (Fig. ​(Fig.11). Therefore, the in vitro susceptibility profile of BCC to doripenem is comparable to those of BCC to meropenem, and it can be an effective therapy for BCC strains resistant to meropenem.

Genomics Reveals a Unique Clone of Burkholderia cenocepacia Harboring an Actively Excising Novel Genomic Island

Burkholderia cenocepacia is a clinically dominant form among the other virulent species of Burkholderia cepacia complex (Bcc). In the present study, we sequenced and analyzed the genomes of seven nosocomial Bcc isolates, five of which were isolated from the bloodstream infections and two isolates were recovered from the hospital setting during the surveillance. Genome-based species identification of the Bcc isolates using a type strain explicitly identified the species as B. cenocepacia. Moreover, single nucleotide polymorphism analysis revealed that the six isolates were clonal and phylogenetically distinct from the other B. cenocepacia. Comparative genomics distinctly revealed the larger genome size of six clonal isolates as well as the presence of a novel 107 kb genomic island named as BcenGI15, which encodes putative pathogenicity-associated genes. We have shown that the BcenGI15 has an ability to actively excise from the genome and forming an extrachromosomal circular form suggesting its mobile nature. Surprisingly, a homolog of BcenGI15 was also present in the genome of a clinical isolate named Burkholderia pseudomallei strain EY1. This novel genetic element is present only in the variants of B. cenocepacia and B. pseudomallei isolates suggesting its interspecies existence in the main pathogenic species of the genus Burkholderia. In conclusion, the whole genome analysis of the genomically distinct B. cenocepacia clinical isolates has advanced our understanding of the epidemiology and evolution of this important nosocomial pathogen as well as its relatives.

MALDI-TOF mass spectrometry: An emerging tool for unequivocal identification of non-fermenting Gram-negative bacilli

Background & objectives: Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) has been instrumental in revolutionizing microbiological identification, especially in high-throughput laboratories. It has enabled the identification of organisms like non-fermenting Gram-negative bacilli (NFGNB), which has been a challenging task using conventional methods alone. In this study an attempt was made to validate MALDI-TOF MS for the identification of clinical isolates of each of the three most common NFGNB, other than Pseudomonas spp., taking molecular methods as the gold standard. Methods: One hundred and fifty clinical isolates of NFGNB, confirmed by molecular methods such as Acinetobacter baumannii[oxa-51 polymerase chain reaction (PCR)], Burkholderia cepacia complex (expanded multilocus sequence typing) and Stenotrophomonas maltophilia (species-specific PCR), were taken. Isolated colonies from fresh cultures of all 150 isolates were smeared onto ground steel plate, with and without formic acid extraction step. The identification was carried out using MALDI-TOF MS Biotyper database. Results: A concordance of 100 and 73.33 per cent was found between the molecular techniques and MALDI-TOF MS system in the identification of these isolates up to genus and species levels, respectively. Using a cut-off of 1.9 for reliable identification, rate of species identification rose to 82.66 per cent. Principal component analysis dendrogram and cluster analysis further increased discrimination of isolates. Interpretation & conclusions: Our findings showed MALDI-TOF MS-based identification of NFGNB as a good, robust method for high-throughput laboratories.

Antimicrobial susceptibility pattern of Burkholderia cepacia complex & Stenotrophomonas maltophilia over six years (2007-2012)

Sir, Non-fermenting Gram-negative bacteria (NFGNB) are a threat to the health care community because these cause opportunistic infections in critically ill or immunocompromised patients. Following Acinetobacter species and Pseudomonas aeruginosa, Burkholderia cepacia complex (Bcc) and Stenotrophomonas maltophilia are the third and fourth common NFGNB among the positive blood cultures at a tertiary care institute in north India1,2. The treatment of infections caused by these organisms is challenging because of high intrinsic and acquired resistance to all commonly used antibiotics including the antipseudomonal drugs3. The increasing incidence of infections by these organisms along with the rising drug resistance warrants a close monitoring of the antimicrobial susceptibility of these organisms. We present the analysis of the antimicrobial susceptibility profiles from March 2007 to December 2012 of 186 Bcc isolates (63 isolates from 2007 to 2009, 89 isolates from 2010 to 2011 and 34 isolates in 2012) and 125 S. maltophilia isolates (38 isolates from 2007 to 2009, 54 isolates from 2010 to 2011 and 33 isolates in 2012) obtained from various clinical specimens (blood, cerebrospinal fluid, sputum, endotracheal aspirate, bronchoalveolar lavage and pus) at the Postgraduate Institute of Medical Education and Research (PGIMER), Chandigarh, India. All the NFGNB isolates were identified by conventional biochemical reactions. Gram-negative, motile, NFGNB were identified further by the use of oxidase test, triple sugar iron agar with lead acetate paper strip and decarboxylase tests4. Molecular identification and typing of Bcc were done by recA polymerase chain reaction-restriction fragment length polymorphism (recA PCR-RFLP)4. Drug susceptibility was tested by Kirby-Bauer disk diffusion test (DD)5 against co-trimoxazole (TMP-SMX, 1.25 µg/23.75 µg), ceftazidime (30 µg), tetracycline (30 µg)/minocycline (30 µg), levofloxacin (5 µg) for S. maltophilia and additionally against meropenem (10 µg) for Bcc following Clinical Laboratory Standards Institute (CLSI) guidelines6. The minimum inhibitory concentrations (MIC) of selected number of isolates were determined by agar dilution method as per CLSI guidelines6 against minocycline (sensitive, S≤4 and resistant R≥16 μg/ml), levofloxacin (S≤2 & R≥8 μg/ml), ceftazidime (S≤8 & R≥32 μg/ml), chloramphenicol (S≤8 & R≥32 μg/ml) for Bcc, and minocycline (S≤4 & R≥16 μg/ml), levofloxacin (S≤2 & R≥8 μg/ml), co-trimoxazole (S≤2/38 & R≥4/76 μg/ml), chloramphenicol (S≤8 & R≥32 μg/ml), ceftazidime (S≤8 & R≥32 μg/ml) for S. maltophilia. The proportional data were analyzed by Z-test for proportions. In 2012, by DD 100 per cent of the Bcc isolates were susceptible to minocycline, 71 per cent to co-trimoxazole, 71 per cent to meropenem and 59 per cent to ceftazidime. A significant decrease in susceptibility of Bcc for ceftazidime has been observed in 2012 (59%) when compared with previous years (83 and 85%, P<0.001). There was no significant decrease in susceptibility to co-trimoxazole and meropenem over the years (Fig. 1). Fig. 1 Percentage susceptibility of Bcc isolates by disc diffusion. ***P<0.001 compared with values in 2007-2009 and 2010-2011. In 2012, by DD 100 per cent of the S. maltophilia isolates were susceptible to minocycline, 100 per cent to levofloxacin, 83 per cent to co-trimoxazole and 25 per cent to ceftazidime. The sensitivity of S. maltophilia to co-trimoxazole (70 to 91%) and levofloxacin (83 to 100%) showed significant variation in contrast to ceftazidime which remained low over the years (25 to 40%). The year-wise susceptibility of the S. maltophilia isolates is shown in Fig. 2. Fig. 2 Percentage susceptibility of Stenotrophomonas maltophilia isolates by disc diffusion. All Bcc and S. maltophilia isolates were sensitive to minocycline though the susceptibility to tetracycline tested in the previous years was less indicating minocycline as a better drug. The MIC values were calculated for 30 S. maltophilia and 60 Bcc isolates. In case of S. maltophilia the percentage of isolates which had MICs within the susceptible range was as follows: 100 per cent for minocycline and levofloxacin, 97 per cent for co-trimoxazole, 64 per cent for chloramphenicol and 50 per cent for ceftazidime. In case of Bcc the percentage of isolates which had MICs within the susceptible range was as follows: 75 per cent for minocycline, 27 per cent for levofloxacin and ceftazidime, 13 per cent for chloramphenicol. Co-trimoxazole had most consistent antimicrobial activity against both Bcc and S. maltophilia. In a study carried out on NFGNB isolates collected globally7,8,9, co-trimoxazole was found to be the most active antibiotic tested against both of these organisms. Both the organisms were highly susceptible to minocycline (majority tested had MIC ≤ 4 µg/ml). S. maltophilia isolates showed lower MIC than the Bcc isolates. Similar variations in the antimicrobial susceptibility between species or between different genera among NFGNB has been documented in a previous study7. Based on the findings of the present and the previous studies7, levofloxacin can be considered as a good alternative in treating S. maltophilia infections. These findings emphasize the importance of correct identification of these organisms and their antimicrobial susceptibility as Bcc is known to be intrinsically resistant to polymyxin and S. maltophilia to carbapenems and both are resistant to aminoglycosides. Routine monitoring of antimicrobial susceptibility pattern of these organisms is mandatory for future policy in the management of such infections.

Hospital Infection Prevention Program

Health care saves lives and has brought unprecedented benefits to generations of patients and their families. However, it also carries risks. Healthcare-associated infection (HCAI) is sometimes the unfortunate consequence of modern medicine: new diagnostic and therapeutic procedures, new treatments for advanced malignancy, organ transplantation, and intensive care are associated with an increased risk of infection [1, 2].