Clonal evolution multi-drug resistant Acinetobacter baumannii by pulsed-field gel electrophoresis

Background: Acinetobacter baumannii is usually multi-drug resistant (MDR), including third generation cephalosporins, amino glycosides and fluoroquinolone. Resistance to these antibiotics is mediated by multiple factors such as: lactamases, efflux pumps and other mechanisms of resistance. Pulsed-field gel electrophoresis (PFGE) was then used to investigate the genetic relationships among the MDR isolates. Aim: The aim of this study was to determine MDR isolates and the existence of OXAs genes among MDR isolates of A. baumannii collected from Kermanshah hospitals in west of Iran. Materials and Methods: Forty-two MDR A. baumannii were collected from patients at Kermanshah hospitals. The isolates were identified by biochemical tests and API 20NE kit. The susceptibility to different antibiotics by disk diffusion method was determined. Polymerase chain reaction (PCR) was performed for detection of bla_OXA-23-like, bla_OXA-24-like, bla_OXA-51-like and bla_OXA-58-like betalactamase genes in isolates and clonal relatedness was done by PFGE (with the restriction enzyme ApaI) and patterns analyzed by Bionumeric software. Results: This study showed high resistant to ciprofloxacin, piperacillin, ceftazidime and also resistant to other anti-microbial agents and more spread blaOXA-23-like gene (93%) in MDR isolate. The PFGE method obtained six clones: A (10), B (9), C (5), D (4), E (11) and F (3) that clone E was outbreak and dominant in different wards of hospitals studied. Conclusion: An isolate from the emergency ward of these hospitals had indistinguishable isolates PFGE profile and similar resistance profile to isolates from intensive care unit (ICU), suggesting likely transmission from ICU to emergency via patient or hospital staff contact.     

An Outbreak of Burkholderia cepacia Complex in the Paediatric Unit of a Tertiary Care Hospital

Introduction: Burkholderia cepacia complex (Bcc) has emerged as a serious nosocomial pathogen worldwide especially in patients with indwelling catheters and cystic fibrosis. Bcc is a common contaminant of pharmaceutical products. We describe an outbreak of Bcc bacteraemia amongst children admitted in Paediatric Intensive Care Unit (PICU) and paediatric ward at a tertiary care hospital, Mumbai, in Western India. Materials and Methods: Blood culture samples from paediatric patients yielded growth of non-fermenting, oxidase positive, motile, Gram negative bacilli (NFGNB) (76/909) over a period of 8 months. Based on conventional biochemical tests and antimicrobial susceptibility testing, these isolates were provisionally identified as Bcc. The increased, repeated and continued isolation of Bcc alerted the possibility of an outbreak confined to PICU and paediatric ward. Active surveillance was undertaken to trace the source and contain the outbreak. Isolates were subjected to recA polymerase chain reaction (PCR) and Expanded multilocus sequence typing (EMLST). Results: Surveillance revealed the presence of Bcc on the upper surface of rubber stopper of sealed multidose amikacin vials. Isolates from blood culture and rubber stoppers were confirmed as Bcc by recA PCR. EMLST revealed that these isolates shared an identical novel sequence type 824 proving clonality. Timely interventions instituted led to control of the outbreak. Conclusion: This study highlights the importance of identification and molecular characterization of Bcc to establish its role in infection and outbreak.     

Candida albicans outbreak associated with total parenteral nutrition in the neonatal unit

Background: The most frequently isolated fungi in patients using TPN belongs to the Candida genus. Various infections including venous catheter infections, fungemia, endocarditis and ophthalmitis may be encountered. Objective: Upon growth of Candida in the blood cultures from the pediatric (neonatal) unit of our hospital, a surveillance was performed in this unit and involving the health care workers. Clonal relationships of the isolates were investigated with molecular tests. Methods: Blood samples obtained from the patients in pediatric neonatal unit were studied with automatized blood culture [BacT/Alert (Bio Mérioux, France)]. Yeast isolates from environmental surveillance cultures (TPN solutions, hands of healthcare personnel, étagère, etc) and patients were identified as C. albicans with conventional methods and ID 32 C and ATB^TM Fungus 3 (Biomerieux, France) kits. Clonal similarity was determined by using AP-PCR as initial method and we have also typified all strains by the method of REP-PCR (diversilab system,bioMérieux). Finally; Pulsed Field Gel Electrophoresis (PFGE) was used for confirmation. Results: C. albicans was isolated in blood cultures of seven patients. Similar antifungal susceptibility patterns were observed in all isolates. AP-PCR and REP-PCR showed that the C. albicans isolates grown in the TPN solution and from the patients’ blood cultures were clonally same strains. PFGE analysis further confirmed this clonality. Conclusion: According to results of the molecular methods, we thought that a C. albicans outbreak had occurred in the neonatal pediatric unit, due to contamination of TPN solution.     

Discriminatory Power of Three Typing Techniques in Determining Relatedness of Nosocomial Klebsiella pneumoniae Isolates from a Tertiary Hospital in India

Purpose: The purpose of this study was to evaluate the discriminatory power of two DNA-based technique and a protein-based technique for the typing of nosocomial isolates of Klebsiella pneumoniae. A second objective was to determine the antimicrobial susceptibility pattern and characterise the presence of genes encoding extended-spectrum beta-lactamases (ESBLs) and carbapenemases. Materials and Methods: Forty-six K. pneumoniae isolates from patients with bloodstream infections at a tertiary care hospital in India between December 2014 and December 2015 were studied. All isolates were typed using enterobacterial repetitive intergenic consensus sequence-polymerase chain reaction (ERIC-PCR), randomly amplified polymorphic DNA (RAPD) analysis and matrix-assisted laser desorption ionisation time-of-flight (MALDI-TOF) mass spectrometry. Antimicrobial susceptibility profiles and ESBLs were detected using the BD Phoenix system. The types of ESBL and carbapenemase genes present were detected using PCR. Results: Isolates were subtyped into 31, 30 and 33 distinct genotypes by ERIC-PCR, RAPD and MALDI-TOF, respectively. Several isolates displaying identical DNA fingerprints were binned into different branches based on their proteomic fingerprint. Antimicrobial susceptibility tests revealed that 33/46 strains were multidrug resistant (MDR); a majority of the strains (83%) were sensitive to colistin. PCR-based analysis demonstrated 19 strains to harbour two or more ESBL and carbapenemase genes. Conclusion: ERIC-PCR was the most reproducible method for typing K. pneumoniae isolates and could not be substituted by MALDI-TOF for clonality analysis. A high degree of genetic diversity and the presence of MDR genes highlight the challenges in treating K. pneumoniae-associated infections.     

Validation of use of whole-cell repetitive extragenic palindromic sequence-based PCR (REP-PCR) for typing strains belonging to the Acinetobacter calcoaceticus-Acinetobacter baumannii complex and application of the method to the investigation of a hospital outbreak.

Acinetobacter spp. are being reported with increasing frequency as causes of nosocomial infection. In order to identify reservoirs of infection as quickly as possible, a rapid typing method that can differentiate epidemic strains from environmental and nonepidemic strains is needed. In 1993, a cluster of Acinetobacter baumannii isolates from five patients in the adult intensive therapy unit of our tertiary-care teaching hospital led us to develop and optimize a rapid repetitive extragenic palindromic sequence-based PCR (REP-PCR) typing protocol for members of the Acinetobacter calcoaceticus-A. baumannii complex that uses boiled colonies and consensus primers aimed at repetitive extragenic palindromic sequences. Four of the five patient isolates gave the same REP-PCR typing pattern as isolates of A. baumannii obtained from the temperature probe of a Bennett humidifier; the fifth isolate had a unique profile. Disinfection of the probe with 70% ethanol, as recommended by the manufacturer, proved ineffective, as A. baumannii with the same REP-PCR pattern was isolated from it 10 days after cleaning, necessitating a change in our decontamination procedure. Results obtained with REP-PCR were subsequently confirmed by ribotyping. To evaluate the discriminatory power (D) of REP-PCR for typing members of the A. calcoaceticus-A. baumannii complex, compared with that of ribotyping, we have applied both methods to a collection of 85 strains that included representatives of six DNA groups within the complex. Ribotyping using EcoRI digests yielded 53 patterns (D = 0.98), whereas 68 different REP-PCR patterns were observed (D = 0.99). By computer-assisted analysis of gel images, 74 patterns were observed with REP-PCR (D = 1.0). Overall, REP-PCR typing proved to be slightly more discriminatory than ribotyping. Our results indicate that REP-PCR typing used boiled colonies is a simple, rapid, and effective means of typing members of the A. calcoaceticus-A. baumannii complex.     

Incidence of Ventilator-associated Pneumonia and Impact of Multidrug-Resistant Infections on Patient’s Outcome: Experience at an Apex Trauma Centre in North India

Introduction: Ventilator-associated pneumonia (VAP) remains one of the most common nosocomial infections in the Intensive Care Unit. In the face of extremely high rates of antimicrobial resistance, it is essential to gauge the clinical significance of isolation of multidrug-resistant (MDR) pathogens from clinical samples. This study details the trend of VAP and the clinical significance of isolation of MDR pathogens from respiratory samples at an Indian tertiary care hospital. Methods: The study was conducted over a 5-year period. VAP was diagnosed on the basis of centres for disease control and prevention criteria. The trend in the rates was compared with preventive measures. Phenotypic and genotypic resistance to beta-lactamases was determined using standard methods. The correlation of isolation of a multi-resistant pathogen with the clinical outcome, length of stay and cost of antimicrobial was ascertained. A clone of Acinetobacter baumannii identified through multilocus sequence typing was used to answer the question of whether resistant bugs always have a fatal outcome. Results: The total ventilator days (VDs) for these patients amounted to 36,278. A total of 433 episodes of VAP occurred during the study, amounting to an overall VAP rate of 11.9/1000 VDs. There was a decline in the rates of VAP over the 5-year period, due to intensive surveillance and preventive activities. A. baumannii (54%) was the most common pathogen, followed by Pseudomonas aeruginosa (21%). A high rate of MDR was seen, with the presence of extended-spectrum beta-lactamases, AmpC and carbapenemase genes. The presence of MDR was not always associated with a fatal outcome. Conclusions: Isolation of MDR pathogens from bronchoalveolar lavage does not always adversely affect the outcome of patients. It requires an interdisciplinary team of clinical microbiologists, physicians and hospital infection control nurses, to collectively manage these patients.     

Nosocomial infections due to Acinetobacter species: Clinical findings, risk and prognostic factors

Purpose: Nosocomial infections caused by Acinetobacter species is of increasing concern in critically ill patients, and the risk factors for this infection are not well established. The present investigation was done to determine incidence of nosocomial Acinetobacter infections. Our study retrospectively attempts to find risk and prognostic factors for the nosocomial acquisition of Acinetobacter infection. Methods: The medical records of 43 patients with Acinetobacter infection during two-year period (Oct1998-Oct2000) were reviewed to find the factors involved in the nosocomial acquisition of Acinetobacter. Acinetobacter isolates that were obtained from these patients were phenotypically typed using carbon assimilation tests. Antimicrobial susceptibility testing results were noted from the microbiology records. Results: Acinetobacter baumannii accounted for 41.8% (n=18) of all the infections. By multivariate logistic regression analysis, only resistant antibiotype {(Ceftazidime- OR, 7.13 [95% CI, 1 to 46]; P= 0.044); (Cefotaxime- OR, 6.09 [CI, 0.87 to 30]; P = 0.045)} and mechanical ventilation (OR, 5.84 [CI, 0.83 to 31]; P = 0.05) were found to be potential independent risk factors for mortality. Overall mortality rate was 33%. Conclusions: Most of A. baumannii isolates were multidrug resistant in our set up and infections due to them were associated with high mortality. Prevention of Multiple drug resistant (MDR) A. baumannii infections was achieved after discontinuation of cefotaxime in ICU. Infection with resistant clones and mechanical ventilation were found to be potential independent risk factors for mortality.     

Tracking Nosocomial Klebsiella pneumoniae Infections and Outbreaks by Whole-Genome Analysis: Small-Scale Italian Scenario within a Single Hospital

Multidrug-resistant (MDR) Klebsiella pneumoniae is one of the most important causes of nosocomial infections worldwide. After the spread of strains resistant to beta-lactams at the end of the previous century, the diffusion of isolates resistant to carbapenems and colistin is now reducing treatment options and the containment of infections. Carbapenem-resistant K. pneumoniae strains have spread rapidly among Italian hospitals, with four subclades of pandemic clonal group 258 (CG258). Here we show that a single Italian hospital has been invaded by three of these subclades within 27 months, thus replicating on a small scale the “Italian scenario.” We identified a single clone responsible for an epidemic outbreak involving seven patients, and we reconstructed its star-like pattern of diffusion within the intensive care unit. This epidemiological picture was obtained through phylogenomic analysis of 16 carbapenem-resistant K. pneumoniae isolates collected in the hospital during a 27-month period, which were added to a database of 319 genomes representing the available global diversity of K. pneumoniae strains. Phenotypic and molecular assays did not reveal virulence or resistance determinants specific for the outbreak isolates. Other factors, rather than selective advantages, might have caused the outbreak. Finally, analyses allowed us to identify a major subclade of CG258 composed of strains bearing the yersiniabactin virulence factor. Our work demonstrates how the use of combined phenotypic, molecular, and whole-genome sequencing techniques can help to identify quickly and to characterize accurately the spread of MDR pathogens.     

Monoclonal outbreak of Ralstonia solanacearum catheter-related bloodstream infection associated with contaminated package of normal saline solution in a tertiary care hospital

Background/aim Ralstonia solanacearum is a very rare cause of infection in humans. There is no described nosocomial outbreak due to R. solanacearum so far. We determined R. solanacearum as the source of catheter-related bloodstream infection (CRBSI) outbreak.Materials and methods This outbreak analysis was carried out in a 1000-bed tertiary care university hospital in Turkey. The outbreak analysis included hematology, oncology, nephrology, gastroenterology wards, emergency department, and intensive care units. The first case with R. solanacearum CRBSI was detected on May 20, 2019 and R. solanacearum was isolated in catheter blood cultures in 34 patients until October 3, 2019Results Standard outbreak analysis procedures were applied. Culture samples were taken from the fluids administered via catheters. The cultures did not yield any bacteria. As a result of the investigation in storage area, it was found that there were leaks, air bubbles, and water drops inside the packaging of saline solutions. R. solanacearum was yielded in the cultures obtained from the surface of saline bags and the inner sides of plastic packings. To validate our hypothesis, a clonal analysis was performed using arbitrarily primed-PCR method and Sanger sequencing of the 16S rRNA gene for identification among isolates. All R. solanacearum isolates were monoclonal and identical.ConclusionThis is the first outbreak of R. solanacearum CRBSI described in a hospital setting. The source of the outbreak was a contamination in the surface of saline bags and the inner sides of plastic packings. Efficacy of an active surveillance system, accurate and rapid conduction of microbiological identification are essential for outbreak management.     

The use of E-test for the drug susceptibility testing of Mycobacterium tuberculosis – A solution or an illusion?

Aim: To evaluate E-test as a tool for rapid determination of drug susceptibility against the conventional LJ method focusing on reliability, expense, ease of standardization and performance of the technique in low resource settings. Materials and Methods: A total of 74 clinical isolates (2004-2005) of Mycobacterium tuberculosis were tested using E-test for susceptibility to streptomycin (STM), isoniazid (INH), rifampicin (RIF) and ethambutol (EMB) by E-strip and LJ (LJPM) proportion methods. Results: The LJPM method, the gold standard, detected resistance against STM in 16.2%, INH in 40.5%, RIF in 18.9% and EMB in 27% cases. In comparison, the resistance values showed by E-test was 66.67% for STM, 57.14% for INH 71.43% for RIF and 80% for EMB. The susceptible correlation was 90.32% for STM, 73.91% for INH, 93.33% for RIF and 59.26% for EMB. E-test correctly identified only eight of the 12 (66.6%) MDR isolates and wrongly identified four isolates which were not MDR. The overall agreement between the two methods was only 48.6%. Resistant isolates showed false positive resistance observed while using E-strip towards all the drugs. Conclusion: E-strips are not quite feasible as a replacement for LJ-proportion method on a large scale due to high risk of cross contamination, laboratory infection, expense associated with it and high false positive resistance observed to all first line drugs. However, the good correlation observed for RIF between the two methods indicates that E-test could contribute to the role in rapid screening of MDR TB isolates as rifampicin mutations are invariably observed in MDR TB isolates.     

An outbreak of CTX-M-15-producing Klebsiella pneumoniae isolates in an intensive care unit of a teaching hospital in Kuwait

Objective: This study reports an outbreak of Klebsiella pneumoniae infections in 14 patients during a 2-month period (August–September, 2008) in the intensive care unit (ICU) of a teaching hospital in Kuwait. Materials and Methods: The clinical sources were blood (9), urine (3) and respiratory secretions (2) identified by the automated VITEK-2 ID System. Susceptibility testing was performed by the E-test method. Extended-spectrum β-lactamase (ESBL) production was assessed using the ESBL E-test and confirmed by PCR. Carriage of bla genes was determined by PCR and sequence analysis. The transferability of resistance phenotypes was demonstrated by conjugation experiments and clonal relatedness was determined by PFGE. Results: The isolates were susceptible to imipenem, meropenem, and tigecycline and produced ESBL. All isolates yielded an amplicon of 499 bp with universal consensus primers (MA primers). DNA sequence analysis showed that they all harboured bla_CTX-M-15and bla_TEM-1genes. The environmental isolate obtained from a suction machine was also CTX-M-15/TEM-1 producer. The resistance phenotypes were transferrable to the Escherichia coli J53^r strain. PFGE, revealed two clones, A and B, related with a Dice coefficient of >94.1%. A mortality rate of 21.4% was recorded. Conclusion: The outbreak was contained by robust and aggressive infection control measures. This study highlights the first outbreak of CTX-M-15-producing K. pneumoniae associated with high mortality in an adult medical ICU in Kuwait.     

Ultrasound Gel as a Source of Hospital Outbreaks: Indian Experience and Literature Review

Purpose: Hospital outbreaks are observed increasingly worldwide with various organisms from different sources such as contaminated ultrasound gel, intravenous (IV) fluids and IV medications. Among these, ultrasound gel is one of the most commonly reported sources for Burkholderia cepacia complex (Bcc) outbreaks. In this study, we describe our experience on investigation and the management of Bcc bacteraemia outbreak due to contaminated ultrasound gel from a tertiary care centre, South India. Materials and Methods: Over a 10-day period in October 2016, seven children in our Paediatric intensive care unit (ICU) were found to have bacteraemia with Bcc isolated from their blood culture. Repeated isolation of the same organism with similar antimicrobial susceptibility pattern over a short incubation period from the same location, confirmed the outbreak. An active outbreak investigation, including environmental surveillance, was carried out to find the source and control the outbreak. Isolates were subjected to multi-locus sequence typing (MLST) and global eBURST (goeBURST) analysis. Results: Environmental surveillance revealed contaminated ultrasound gel as the source of infection. MLST and goeBURST analysis confirmed that the outbreak was caused by a novel sequence type 1362 with the same clonal complex CC517. The outbreak was controlled by stringent infection control measures, withdrawal of contaminated ultrasound gel from regular usage and implementing the practice of using ultrasonogram (USG) probe cover for USG screening and guided procedures. Conclusion: This report highlights the importance of early identification of an outbreak, prompt response of the ICU and infection control teams, sound environmental and epidemiological surveillance methods to identify the source and stringent infection control measures to control the outbreak. Contaminated ultrasound gel can be a potential source for healthcare-associated infection, which cannot be overlooked.     

Assessment of trends of ofloxacin resistance in Mycobacterium tuberculosis

Purpose: Ofloxacin (OFX) is one of the potent fluoroquinolone (FQ) recommended to treat MDR-TB. Over a decade, the preexposure of this drug for the treatment of other bacterial infections has resulted in acquisition of FQ resistance among Mycobacterium tuberculosis strains. Considering this possibility, a study was undertaken in a tertiary care center in the capital city (India) to assess the drug resistance trends of OFX among susceptible and multidrug resistant (MDR) strains of M. tuberculosis. Materials and Methods: A total of 102 M. tuberculosis isolates (47 susceptible to fi rst-line drugs and 55 MDR isolates) were screened for susceptibility testing of OFX with a critical concentration of 2 μg/ml by Lowenstein Jensen (LJ) proportion method. Results: The results showed 40 (85.1%) isolates among 47 susceptible isolates and 34 (61.8%) isolates among 55 MDR isolates, were found to be susceptible to OFX. Fisher’s exact test showed significant P-value (0.0136) demonstrating 1.377 fold (95% confidence interval) increased risk to become resistant to OFX than susceptible isolates. These finding shows decreased OFX susceptibility is not only limited to MDR isolates but also increasingly seen in susceptible strains as a result of drug abuse. Conclusions: Our finding were not alarming, but highlights the general risk of acquiring resistance to OFX, jeopardizing the potential for these drugs to be used as second-line anti-TB agents in the management of drug-resistant TB and creating incurable TB strains.     

Incidence,risk factors,microbiology of venous catheter associated bloodstream infections - A prospective study from a tertiary care hospital

Purpose: Central venous catheters (CVCs) though indispensable in current medical and intensive care treatment, also puts patients at risk of catheter related infection (CRI) resulting in increased morbidity and mortality. We analysed the incidence, risk factors, bacteriological profile and antimicrobial susceptibility pattern of the isolates in central venous catheter associated bloodstream infection (CVC-BSI) in the intensive care unit (ICU) patients and studied the formation of biofilm in CVCs. Materials and Methods: The following case control study included 115 patients with CVC in situ. Quantitative blood cultures (QBC) and catheter tip cultures were performed for the diagnoses. Direct catheter staining was done for an early diagnosis by acridine orange (AO) and Gram staining methods. Biofilm production in catheters was detected by ‘tissue culture plate’ (TCP) method. The results were analysed using the computer-based program statistical package for the social sciences (SPSS). Results: In 25/115 patients, definite diagnosis of CVC-BSI was made. The mean age was 48.44 ± 17.34 years (cases) vs 40.10 ± 18.24 years (controls) and the mean duration of catheterisation was 25.72 ± 8.73 days (cases) vs 11.89 ± 6.38 days (controls). Local signs of infection (erythema, tenderness and oozing) were found more significantly in CVC-BSI cases. The AO staining was more sensitive and Gram staining of catheters showed higher specificity. Staphylococcus aureus followed by Pseudomonas aeruginosa and non-albicans Candida were common CVC-BSI pathogens. Multidrug-resistant (MDR) strains were isolated in bacterial agents of CVC-BSI. Non-albicans Candida and Enterococcus faecalis showed strong biofilm production. Conclusion: The incidence of CVC-BSI was 21.73% and the rate was 14.59 per 1000 catheter days. Prolonged ICU stay and longer catheterisation were major risk factors. S. aureus was isolated most commonly in CVC-BSI cases. The menace of multidrug resistance and biofilm formation in CVCs is associated with CVC-BSI.     

Outbreak of Burkholderia cepacia complex bacteremia in a chemotherapy day care unit due to intrinsic contamination of an antiemetic drug

Background: In the end of 2009, a large number of patients with cancer undergoing chemotherapy at the day care unit of a private hospital in Mumbai, India developed Burkholderia cepacia complex (BCC) blood stream infection (BSI). Objective: The objectives were to identify the source of the outbreak and terminate the outbreak as rapidly as possible. Materials and Methods: All infection control protocols and processes were reviewed. Intensive training was started for all nursing staff involved in patient care. Cultures were sent from the environment (surfaces, water, air), intravenous fluids, disinfectants and antiseptics and opened/unopened medication. Results: A total of 13 patients with cancer with tunneled catheters were affected with BCC BSI. The isolates were of similar antimicrobial sensitivity. No significant breach of infection control protocols could be identified. Cultures from the prepared intravenous medication bags grew BCC. Subsequently, culture from unused vials of the antiemetic granisetron grew BCC, whereas those from the unopened IV fluid bag and chemotherapy medication were negative. On review, it was discovered that the outbreak started when a new brand of granisetron was introduced. The result was communicated to the manufacturer and the brand was withdrawn. There were no further cases. Conclusions: This outbreak was thus linked to intrinsic contamination of medication vials. We acknowledge a delay in identifying the source as we were concentrating more on human errors in medication preparation and less on intrinsic contamination. We recommend that in an event of an outbreak, unopened vials be cultured at the outset.     

Molecular Characterisation for Clonality and Transmission Dynamics of an Outbreak of Klebsiella pneumoniae amongst Neonates in a Tertiary Care Centre in South India

Purpose: Sepsis is a significant cause of morbidity and mortality amongst neonates. Klebsiella pneumoniae is a common cause of nosocomial outbreaks causing bacteraemia and having potential of acquiring plasmids enhancing antimicrobial resistance. In the present study, we investigate K. pneumoniae outbreak causing bacteraemia amongst neonates over a span of 2 months. Isolates were characterised for antimicrobial resistance, virulence, molecular typing for clonality and plasmid typing for transmission dynamics, and patient outcome was investigated. Methods: Thirteen isolates of K. pneumoniae were obtained during October–November 2016. Antimicrobial susceptibility testing was performed, and multiplex polymerase chain reaction (PCR) for β-lactamases and PCR for ompK35 and ompK36 were performed. To study hypervirulence, string test and PCR for rmpA and rmpA2 were performed. Multilocus sequence typing and Inc plasmid typing were carried out to study transmission dynamics. Results: Amongst 13 isolates, all isolates harboured bla_SHV and bla_TEM; 12 isolates carried bla_CTX-M-1. ompK35 was present in all, but ompK36 was absent in 12 isolates. Ten isolates belonged to ST48, 6 amongst which contained IncFII (K) plasmid. One isolate each belonged to ST29, ST111 and ST2647 (novel clone). None of the isolates was hypervirulent. Conclusion: Extended-spectrum β-lactamase K. pneumoniae is commonly seen in Indian hospitals and main mechanisms being production of SHV, TEM and CTX-M enzymes as seen in the present study. Outer membrane porins contribute significantly to antimicrobial resistance. Emergence of new clones such as ST2647 implies continuous evolution of the organism and also potential for rapid genetic recombination leading to multidrug resistance. Outbreaks amongst neonates lead to fatal outcome, and stringent hospital infection control is necessary.     

PCR-based DNA fingerprinting (REP-PCR, AP-PCR) and pulsed-field gel electrophoresis characterization of a nosocomial outbreak caused by imipenem- and meropenem-resistant Acinetobacter baumannii

Objective   To demonstrate the usefulness of REP-PCR and AP-PCR on molecular typing of A. baumannii isolates.
Method   From February to November 1997, 29 inpatients at Ramón y Cajal Hospital, Madrid—23 in five intensive care units (ICUs) and six at two different medical departments—were either colonized or infected with imipenem- and meropenem-resistant Acinetobacter baumannii (IMRAB) strains (MICs of 64–256 mg/L). A wide antibiotic multiresistance profile was observed with IMRAB strains, and only tobramycin, sulbactam and colistin displayed valuable activity. For typing IMRAB isolates, repetitive extragenic palindromic sequence-based polymerase chain reaction (REP-PCR) and arbitrary primer sequence-based polymerase chain reaction (AP-PCR) methods were used and compared with pulsed-field gel electrophoresis (PFGE) as reference technique. For comparative purposes, 30 imipenem- and meropenem-susceptible A. baumannii (IMSAB) strains isolated before, during and after the outbreak were included in this study.
Results   The molecular typing results showed that the outbreak was caused by a single IMRAB strain (genotype 1). On the other hand, seven different genotypes were observed in the pre-, at- and post-outbreak strains tested by REP-PCR. Regarding AP-PCR, three of four at-outbreak IMSAB strains were indistinguishable from the IMRAB profile. Thus, with AP-PCR, only six genotypes were obtained, apart from the IMRAB genotype.
Conclusion   Under our experimental conditions, REP-PCR had a higher discriminatory power than AP-PCR, with PFGE as reference technique. The REP-PCR technique is a useful and expeditious method for the epidemiologic characterization of A. baumannii nosocomial outbreaks, the results being comparable to those obtained with the PFGE technique.     

Emergence and control of an outbreak of infections due to Panton-Valentine leukocidin positive,ST22 methicillin-resistant Staphylococcus aureus in a neonatal intensive care unit

Methicillin resistant Staphylococcus aureus (MRSA) infection can cause significant morbidity and mortality in neonates. We investigated a nosocomial MRSA outbreak in a neonatal intensive care unit (NICU), using a novel typing method. Following two fatal cases, in May 2011, a prospective outbreak investigation was conducted, involving neonates, mothers and healthcare workers in a large tertiary NICU in Sydney. MRSA isolates were characterized by antimicrobial susceptibility testing, a multiplex PCR-based reverse line blot (mPCR/RLB) binary typing system and other molecular typing methods. Over 7 months, 14 neonates were colonized with MRSA and six infected: three with superficial lesions and three with life-threatening disease, including the two index cases, who died despite empirical treatment with vancomycin. Isolates from 15 neonates were indistinguishable by RLB typing and identified as a PVL-producing ST22 SCCmec IV MRSA strain, which was resistant to gentamicin and trimethoprim-sulphamethoxazole. The outbreak strain was also isolated from one healthcare worker, one environmental swab and one father, but the source remained obscure. During the same period several different non-multiresistant and multiresistant MRSA strains were isolated from five neonates, five mothers (including two whose infants were colonized with the outbreak strain), one father, three healthcare workers and two environmental swabs. Rapid turnaround time of typing results allowed us to recognize and define the outbreak and implement targeted infection control interventions. PVL-producing ST22 SCCmec IV MRSA appears to be a virulent and highly transmissible pathogen in the NICU, which was difficult to control.     

Bacteriological profile and antibiotic sensitivity pattern of neonatal septicaemia in a rural tertiary care hospital in North India

Background: There is not much published literature on neonatal septicemia available for the Sub-Himalayan region of North India. Hence, we undertook this study to find out the bacteriological profile and antibiotic sensitivity pattern of neonatal septicemia in the neonatal Intensive Care Unit. Material and Methods: Blood cultures were performed for all clinically suspected neonatal septicemia cases for 1-year. Identification of all pathogenic isolates was followed by antibiotic sensitivity testing. Results: We did blood cultures for 450 neonates and 42% were culture positive. Early onset sepsis were 92 (49%) and 96 (51%) were late onset sepsis. Gram-positive isolates were 60% and 40% were Gram-negative. Staphylococcus aureus (40%), coagulase negative Staphylococcus species (16%), non-fermenter group of organisms (NFGOs) (15%), and Klebsiella pneumoniae (10%) were the main isolates. Nasal cannula 101 (54%), birth asphyxia 91 (48%), and prematurity 73 (38%) were the prominent risk factors associated with septicemia. Gram-positive organisms were highly resistant to penicillin (87%) whereas Gram-negative isolates showed high resistance to third generation cephalosporins (53–89%) and aminoglycosides (50–67%). The S. aureus isolates were methicillin-resistant in 41% whereas extended spectrum beta lactamase production was seen in 48% Gram-negative isolates. Conclusion: Our study highlights the recent emergence of Gram-positive organisms as predominant cause of neonatal septicemia in this part of Sub-Himalayan region, along with the review of literature which shows similar results from North India and rest of the world too. Though Gram-negative bacteria still remain the main cause of mortality in neonatal septicemia, we want to dispel the common notion among practitioners that they are the predominant isolates in neonatal septicemia.