Clinical Laboratory Standards Institute Guidelines Research Articles

Molecular Capsular Typing and Multi Locus Sequence Typing of Invasive, Non-invasive and Commensal Streptococcus pneumoniae Isolates from North India

AbstractPurpose: The purpose of this study is to determine the antimicrobial susceptibility pattern, serotype distribution and sequence type (ST) of Streptococcus pneumoniae isolates from invasive and non-invasive infection and correlate it with isolates from commensal nasopharyngeal flora to ascertain their role in infection. Materials and Methods:S. pneumoniae isolates from blood, cerebrospinal fluid, pleural fluid and respiratory secretions (sputum, bronchoalveolar lavage and nasopharyngeal swab/throat swab) were analysed to determine ST, serotype and antimicrobial susceptibility pattern. Serotyping was performed by multiplex polymerase chain reactions as well as by quellung reaction. Antimicrobial susceptibility testing was determined using Kirby Bauer’s disc diffusion method as per the Clinical Laboratory Standards Institute guidelines. Minimum inhibitory concentration was determined using E-test for penicillin. Multilocus sequence typing (MLST) was done to understand genetic relatedness and evolutionary relationship among strains. Results: A total of 125 S. pneumoniae isolates were collected, including 25 from invasive pneumococcal disease, 25 from non-invasive and 75 from nasopharyngeal swab of healthy children (Commensal). Resistance to penicillin, erythromycin, and co-trimoxazole was observed in 14.4%, 12% and 81.6% of the isolates, respectively, by KirbyBauer’s disc diffusion method. Serotype 14 was found to be the most prevalent in invasive and non-invasive isolates, while serotype 6 was the most common in commensal isolates. New STs were found among invasive (ST13826, ST13827), non-invasive (ST13823, ST13824, and ST13961) and commensal (ST13825) isolates. Conclusion: MLST sequence analysis shows that invasive isolates were found to be clustered with non-invasive and commensal isolates. Analysis of MLST suggests the possibility of genetic relatedness and exchange of genetic material between invasive, non-invasive and commensal isolates.

Antimicrobial resistance pattern of bacterial isolates and genetic resistance determinants of carbapenemase producers in bloodstream infections

Introduction: Bloodstream infection (BSI) continues to be a significant cause of disease and death in hospitalized patients worldwide. These are among the most common healthcare-associated infections with a mortality rate of 20%–50%. The emergence of multidrug resistance among the organisms causing BSI is of great concern. Objectives: The study was undertaken in a medical intensive care unit of a tertiary care hospital in Eastern India to evaluate the spectrum of pathogens causing BSI, their antimicrobial resistance patterns and carbapenemase enzyme production using polymerase chain reaction (PCR). Materials and Methods: A total of 550 blood samples from clinically suspected cases of BSIs were studied from July 2016 to June 2018. Blood samples were inoculated and incubated in BacT/ALERT (BioMerieux) system. Identification and antibiotic susceptibility testing was conducted in Vitek-2 (BioMerieux) as per the Clinical Laboratory Standards Institute guidelines. Methicillin-resistant Staphylococcus aureus (MRSA) and β-lactamase production were also noted in the Vitek-2 method. All Gram-negative isolates were studied for carbapenemase production by the genotypic method using PCR. Results: Of 550 samples, 116 samples yielded the growth of various bacterial isolates. Of these, 54 (47%) were Gram-positive organisms and 62 (53%) were Gram-negative organisms. S. aureus was the most common organism isolated followed by Klebsiella pneumoniae. MRSA was observed in 66.7% of S. aureus isolates. Among Gram-negative isolates, 43.5% were found to be β-lactamase producers and 66.1% were carbapenemase producers. Most of the carbapenemase producers were encoded by the OXA gene (58%). Conclusion: This study emphasizes the importance of antibiotic policy and its stringent application, which will eventually help us to control the menacing rise in antimicrobial resistance.

Screening of Methicillin Resistant Staphylococcus aureus (MRSA) from Wounds in Pediatric Patients Visiting Tertiary Care in Hospital

The extent of methicillin-resistant Staphylococcus aureus (MRSA) in children is still unknown. The collected wound pus samples were processed. Identification of S. aureus was done according to standard microbiological procedures as per the clinical laboratory standards institute (CLSI) guidelines (2016). The antibiogram of the isolates was carried out by the Kirby-Bauer disc diffusion technique. MRSA was determined by measuring the zone of inhibition (ZOI) surrounding to cefoxitin disc, with resistance defined as ZOI of ≤ 21 mm. Out of 357 bacterial culture-positive samples, 278 (77.87 %) were S. aureus isolates, among them 102 (36.69%) were found to be MRSA. The percentage of MRSA isolates was found high in male children and inpatients with 61.76 % and 73.52% respectively. All the MRSA isolates were susceptible to gentamicin (79.41%), whereas (91.17%) were resistant to penicillin. The distribution of MRSA in inpatients 75 (73.52%) is higher than that of outpatients 27 (26.74%). This study shows that the MRSA occurrence is prevalent in pediatric patients.

Surveillance of healthcare associated infections at a tertiary care teaching hospital from 2014 to 2016: Prevalence, microbiological spectrum and antibiogram

Background: Healthcare-associated infections have emerged as a significant cause of mortality in the recent years. One of the major concerns in the prevalence of such infections is the development of multidrug-resistant microorganisms. Aim: The present surveillance study was conducted to assess the prevalence, microbiological spectrum, and antimicrobial susceptibility aminoof the microorganisms at a tertiary care hospital. Methods: The study was conducted between May 2014 and December 2016 including 466 patients (255 men and 211 women) diagnosed with healthcare-associated infections. Data related to sex, age, time of admission, type of surgery, and complication were collected. Samples of the pus, urine, and blood were collected and sent for isolation and characterization of microorganisms. Antimicrobial sensitivity was conducted by following the Clinical Laboratory Standards Institute (CLSI) guidelines. Data obtained were recorded in Microsoft Excel and analyzed using R 3.3.1; P < 0.05 indicative of significant differences. Result: Mean age of the patients was 41.01 ± 18.11 years. Prevalence results were highest in surgical site infec-tion (79.18%) and gram-negative organisms (80.92%) were the prevalent causes of infection. Escherichia coli (21.54%), Pseudomonas aeruginosa (15.76%), and Acinetobacter sp. (12.22%) were the predominant organ-isms with maximum sensitivity towards carbapenems, aminoglycosides, and cotrimoxazole, respectively. E. coli was the prevalent producer of AmpC and extended-spectrum beta-lactamases. Conclusion: Reduced sensitivity to the majority of antimicrobials has led to the prevalence of gram-negative organisms in the healthcare-associated infections. However, carbapenems and chloramphenicol along with aminoglycosides remain the effective broad-spectrum antimicrobials. Further studies are required strategies to minimize antibiotic resistance, which can lead to improvement in therapeutic methods. Keywords: Escherichia coli, Carbapenems, Surgical site infection, Lactamases

Acute rise in Multiple Drug Resistance Typhoid fever in Children in Rural Sindh: an Alarming Situation

Objective: Evaluation of the frequency of antimicrobial drug resistance in Salmonella typhiamong children of rural Sindh. Study Design: Cross sectional / observational study Place and Duration: At Department of Pediatric medicine Civil Hospital Mirpurkhas, Sindh from January 2019 to July 2019 Methodology: With approval of ethical committee and informed consent from parents/attendant blood samples (5ml) of 55 children with age between 0 to 15 years are obtained at outpatient department (OPD) or admitted in the ward with suspected enteric fever. The blood samples were used to obtain bacterial culture and antibiotic sensitivity was performed at diagnostic and research laboratory (DRC) of Liaquat University of Medical and Health Sciences (LUMHS). Blood culture and Antibiotic Susceptibility testing was performed according to standard protocol of Clinical Laboratory Standard Institute (CLSI) guidelines. Results: Among study population (n=55), 32 (58.2% were in age group 0 to 5 years, 20(36.4%) in age group of 6 to 10 years while only 3(5.5%) were in the age group of 11 to 15 years.While regarding gender,29(52.7%) were male while 26(36.4%) were females. S. typhi exhibited high resistance against ampicillin (93% n=51) and cotrimazole-trimethoprim (91%n= 50). About 82% (n=46) of culture was observed resistant to ciprofloxacin, whereas 84% (n=46) showed resistance to ceftriaxone, 84% (n=46) were resistant to cefixime. Out of these 55, 45(82%) showed antimicrobial sensitivity to meropenem, 34 (62%) to azithromycin, 10(18%) to ciprofloxacin, 09 (16%) to cefixime and ceftriaxone each, while only 05 patients showed sensitivity to (9%) to cotrimazole-trimethoprim and 04(7%) to Amoxicillin/clavulanic acid. Conclusion: Multiple drug resistance in S. typhiis high alert situation in our setup and needs prompt initiatives to combat.

Detection of SHV and TEM-type Extended spectrum β-lactamase in bacterial isolates recovered from clinical samples of patients attending military hospitals

BackgroundMulti-drug resistant bacterial strains have been increasingly implicated in clinical infections worldwide and beta-lactamase production is one of the commonest mechanisms of resistance in these strains. This study investigated the prevalence of extended spectrum â-lactamase (ESBL)-producing isolates and determined the temoneira (TEM) and sulfhydryl variable (SHV) types implicated in two military hospitals in South-South Nigeria. MethodsThree-hundred and eighty (380) consecutive non-duplicate bacterial isolates (Gram negative bacilli) recovered from clinical samples were identified following standard techniques. Antimicrobial susceptibility tests were performed for each isolate following the Clinical Laboratory Standards Institute guidelines. Bacterial isolates recovered which comprised Escherichia coli, Klebsiella spp, Proteus spp and Pseudomonas aeruginosa were screened for ESBL using a phenotypic method (double disc synergy test). All positive isolates were screened for TEM and SHV genes by PCR method. ResultsSixty-five isolates (17.1%) were ESBL producing using phenotypic method, E. coli showed the highest ESBL prevalence (24.3%). One isolate was SHV positive (1.5%), 8 (12.3%) were TEM positive while 3 (4.6%) isolates harbored both SHV and TEM genes. Fluoroquinolone - ofloxacin showed marked activity against ESBL-producing isolates (90.8%) while the least active were ceftriaxone (9.2%), ceftazidime (3.1%) and ampicillin (1.5%). ConclusionThis study demonstrated that 17.1% of Gram-negative bacilli were ESBL producers. Screening of clinical isolates for ESBL should be implemented. The findings of this study suggest the need for caution in the use of antimicrobial agents in order to curb the incidence of antimicrobial resistance.

Bacteriological profile and antimicrobial resistance patterns of burn wound infections in a tertiary care hospital

BackgroundThe bacterial infections that prevail in the burnt patients continue to be a critical complication in the burnt patients and vary with time and place. Identification of bacterial pathogens with information of their antimicrobial susceptibility of burn wounds can help clinicians to select appropriate medication procedure as in providing them with suitable antibiotic for empirical treatment. MethodsRetrospective study of thirty-one months (Jan 2015 to July 2017) was designed to evaluate bacteria involved in burnt wound infection and its antimicrobial susceptibilities in a Burn Intensive Care Unit of Eastern India. Pus samples were cultured on cysteine Lactose electrolyte deficient agar (Hi-Media, India). Positive bacteria cultures were identified and tested for antimicrobial susceptibility using VITEK®2 (bioMerieux, Durham, NC, USA) and interpreted according to Clinical Laboratory Standards Institute guidelines. ResultsTwo hundred and seventy-two wound swabs from burnt patients were received, out of which 62.8% (n = 185) were revealed as positive for the presence of bacteria. Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii and E. coli were discovered to be the most common organisms in patients. Isolated bacteria were least resistant to TIGECYCLINE and COLISTIN. ConclusionData regarding the incidence of pathogens and their resistance patterns would benefit the clinicians to prescribe appropriate antibiotics, articulating policies for empirical antimicrobial therapy to control the different types of infections.

2030. Impact of an Antimicrobial Stewardship Program on Carbapenem Susceptibility in a National Hospital in Bhutan

BackgroundThe overuse of broad-spectrum antibiotics drives antimicrobial resistance (AMR), and the prevalence of highly-resistant Gram-negative infections is increasing across the world, especially in low- and middle-income countries (LMIC). Carbapenem resistance is of particular concern since these are often the last line agents. Antimicrobial restriction is an antimicrobial stewardship intervention (AMS) that aims to reduce the use of broad-spectrum antibiotics to preserve antimicrobial susceptibility.MethodsThis is retrospective, observational study of antibiotic consumption and prevalence of antibiotic resistance of bacterial isolates from inpatients at Jigme Dorji Wangchuck National Referral Hospital, a 350-bed multi-specialty hospital in Thimphu, Bhutan. Antibiotic consumption and antimicrobial susceptibility were monitored from January 2015 to December 2017 by the pharmacy department and the microbiology lab, respectively. Antibiotic consumption was measured using defined daily doses (DDD) and expressed as DDDs per 1,000 persons per day. The antibiotic susceptibility was determined using the Clinical Laboratory Standards Institute (CLSI) guideline. A hospital AMS program with multidisciplinary team and good hospital managerial/ leadership support were initiated in 2016 and interventions included antimicrobial restrictions, educations, guidelines for use, post prescription review, de-escalation, audit and feedback.ResultsFrom 2015 to 2016, the DDDs of carbapenems and piperacillin–tazobactam (PTZ) increased while ceftriaxone decreased (Figure 1). After the AMS program was implemented in 2016, the annual DDDs of carbapenems decreased while PTZ and ceftriaxone increased. Antimicrobial susceptibility of Klebsiella pneumoniae and Escheriachia coli blood isolates to carbapenems and ceftriaxone increased from 2016 to 2017: 50/61 (82%) vs. 45/49 (92%) and 24/91 (26%) vs. 31/92 (34%), respectively.ConclusionImplementing an AMS program that restricted the use of carbapenems resulted in a decrease in carbapenem use and increased antimicrobial susceptibility for carbapenems and ceftriaxone. AMS interventions can be successful to decrease carbapenem-resistance in LMIC.

1955. Novel Compound Reverses Vancomycin Resistance in Vancomycin-resistant Enterococci (VRE)

Background Enterococcus causes 14% of all hospital-associated infections (HAIs) according to Centers for Disease Control and Prevention (CDC) data. 35.5% of these HAIs are caused by Vancomycin-resistant Enterococci (VRE) including highly fatal bacteremia, surgical site infections, and urinary tract infections. We present a novel synthetic compound, HSD 03-21 that could make VRE completely susceptible to vancomycin in-vitro.MethodsHSD 03-21 was synthesized de novo from a hydroxybenzylidene – indolinone backbone in our laboratory. The minimum inhibitory concentration (MIC) of HSD 03-21 and vancomycin against VRE were determined according to clinical laboratory standards institute (CLSI) guidelines. The standard checkerboard assay was used to determine vancomycin-HSD 03-21 interactions against VRE. Briefly, HSD 03-21 and vancomycin at 10 mg/mL were prepared and diluted serially along the ordinate and abscissa of 96-well microtiter plates, respectively. Bacteria was standardized using the 0.5 McFarland standard, diluted (1:100), aliquoted into respective wells and incubated at 37oC for 18–20 hours. All assays were run in triplicates. The fractional inhibitory concentration (FIC) index was calculated for each combination. The FIC of either agent was calculated as: FIC (vancomycin) = MIC of vancomycin in combination/MIC of vancomycin alone and FIC (HSD 03-21) = MIC of HSD 03-21 in combination/MIC of HSD 03-21 alone. The cumulative FIC index ∑FICI was then calculated as: ∑FIC = FIC(vancomycin) + FIC(HSD 03-21). The calculated ∑FIC indices were interpreted as synergistic if ∑FIC: ≤ 0.5.ResultsThe MIC of vancomycin for VRE faecalis was 256 μg mL−1 while that of HSD 03-21 was 128 μg mL−1. When vancomycin was combined with HSD 03-21 at 8 μg mL−1 (1/16 MIC), there was a reduction in MIC of vancomycin to 0.5 μg mL−1. The combination showed excellent synergy with ∑FIC of 0.06.ConclusionHSD 03-21 reduced the MIC of vancomycin from 256 to 0.5 μg mL−1. This has an immense potential of changing the way we use vancomycin and in the treatment of VRE infections. Translation of this novel compound could save thousands of lives from VRE and the failures and inherent toxicities of current doses of vancomycin. Disclosures All Authors: No reported Disclosures.

Antibiotic resistance pattern of methicillin-resistant <i>Staphylococcus aureus</i> isolated from children under the age of five Years in Anambra State

Neonates are exposed to Staphylococcus aureus shortly after birth and can become colonized quickly after contact with adult skin or the environment. Methicillin-resistant S. aureus (MRSA) constitutes part of the growing global health problem associated with an increasing number of infections and often multidrug resistant in nature which now poses serious therapeutic problems to clinicians. Eighty-three (83) samples were collected which were cultured on appropriate bacteriological media. Bacterial isolates ( S. aureus ) were identified by standard biochemical tests and confirmed using PCR targeting the 16S rRNA. The MRSA was determined using Oxacillin antibiotic disk and confirmed by the presence of MecA gene. Antibiotic susceptibility of the MRSA isolates to eleven antibiotics was performed according to Clinical Laboratory Standard testing Institute (CLSI) guidelines. Out of the 83 individuals tested, 25 yielded S. aureus of which 22 (88.0%) of them were MRSA positive, 10 (45.5%) females and 12 (54.5%) males. The antibiotic resistant pattern of the 22 MRSA isolates showed Quinupristin/Dalfopristin 13 (59.1%), Fusidic acid 6 (27.3%), Linezolid 8 (36.4%), Clindamycin 10 (45.5%), Vancomycin 5(22.7%), Cefepime 3(13.6%), Doxycycline 4(18.2%), Sulphamethoxazole 22(100%), Fosfomycin 7(31.8%), Cephalexin 7(31.8%), Trimethoprim 19(86.4%). The MIC determination for vancomycin from MRSA isolates showed antibiotic concentration of 4.9 μg/ml and 8.7 μg/ml. This work showed that there is high prevalence rate of resistance to many classes of antibiotic warranting continued surveillance and antimicrobial stewardship. Therefore effective antibiotic susceptibility test should be conducted before prescribing an antibiotic to patients, in as much as patients should strictly adhere to antibiotic prescription to mitigate abuse of drugs. Keywords: Methicillin-resistance, antibiotics, prevalence, S. aureus , susceptibility

P3599Minicare high sensitivity troponin: a novel point-of-care tool to improve ACS workflows

Abstract Background Cardiac troponin (cTn) has been broadly accepted as the biomarker of choice in the diagnosis of patients presenting with Acute Coronary Syndrome (ACS). Point-of-care (POC) cTn testing, defined as testing near the patient with rapid availability of results, has attracted a strong interest in the emergency department (ED) setting. It offers the potential to improve workflows, expedite clinical decisions and to reduce the length of stay. Workflows could be further optimized when a first measurement can already be performed by the emergency medical services prior to admission to the hospital. Advances in assay technology have led to high sensitive (HS) cTn assays that have a profound impact on clinical practice, providing early results at presentation and 1 hour after admission which speed up clinical decision-making. Here we evaluate the Minicare HS cTnI POC test under development, which has the potential to combine the benefits of HS-cTnI protocols with a POC workflow. Objective Evaluate the analytical and clinical capability of the Minicare HS-cTnI test under development to meet the criteria‡ for HS cTn of having a 10% CV <99th percentile and a percentage measurable (>LoD) of >50%. Methods The evaluation is based on the Clinical Laboratory Standards Institute (CLSI) guidelines. Li-heparin whole blood and Li-heparin plasma samples were used to establish LoB, LoD, and LoQ, sample matrix comparison and linearity in the low range. The clinical performance for Minicare HS-cTnI was assessed and compared to Abbott Architect high-sensitivity troponin using banked samples from patients with ACS and collected at t=2–4h after admission. The percentage measurable was assessed and compared to Abbott Architect high-sensitivity troponin in a healthy population of 165 individuals. Results With a time to result within 10 minutes, the LoB is determined at 1.0 ng/L and the LoD at 2.1 ng/L. Plasma and whole blood results correlated well and showed comparable results. The 10% and 20% CV LoQ are established at 7.9 ng/L and 3.1 ng/L, well below the anticipated 99th percentile of 26 ng/L. With a percentage measurable of 77%, the criteria for a HS-cTnI assay are met in a POC device. A sensitivity of 93% and an NPV of 99% are found for Minicare HS-cTnI for a t=2–4h protocol vs 90% and 98% for Abbott Architect high-sensitivity troponin. In this study the area under the curve is 0.98 for Minicare HS-cTnI and 0.97 for Architect HS-cTnI. ROC curve t=2-4 h Conclusions With HS-cTnI capability on the Minicare platform, we show the potential to support a 0/1 h sampling protocol, combined with the speed of a POC workflow. This may enable even more rapid and safe rule-out of patients with ACS.

Prevalence and Susceptibility Analysis of Gram Negative Pathogens in Super Specialty Tertiary Care Centers, Pune in India from January 2018- January 2019

Background and Objective: The encumbrance of antimicrobial resistance worldwide is substantive and likely to rise without any appropriate treatment which has enhanced the urge for the development of either new antibiotics or adjuvant therapy with antibiotics. Thus, we aimed to study a comparative antibiogram pattern of 758 clinical isolates collected from Pawana Hospital and Accord SDH, Pune (India), towards Elores (a novel antibiotic and adjutant entity of ceftriaxone, sulbactam and disodium edetate) and other antibiotics (imipenem, meropenem and piperacillin + tazobactam).
 Methods: The clinical samples collected from outpatients and inpatients during a period of one year (January, 2018 to January, 2019), from Pawana Hospital and Accord SDH, Pune (India) and were further subjected to bacterial identification. Antibiotic susceptibility testing was executed in accordance with the recommendations of Clinical Laboratory Standards Institute (CLSI) guidelines.
 Results: Out of 758 collected samples, urine samples contributed 69.41 and 37.63% among Enterobacteriaceae and Non-Enterobacteriaceae followed by pus (20.25 and 19.35%) and sputum (4.2 and 19.89%) while <4% and <12% Enterobacteriaceae and Non-Enterobacteriaceae isolates were collected from rest of the specimen. E. coli were found most prevalent (50.13%) along with 19.13 and 16.09% prevalence of Pseudomonas spp. and Klebsiella spp. whereas rest of the pathogens were <7% present. The antibacterial activity of Elores (87.80%) was observed superior to carbapenem drugs (meropenem; 62.67% and imipenem; 60.95%) and far better to piperacillin + tazobactam (48.42%) against 758 clinical pathogens. Antibiogram profile depicted Elores as most susceptible (95.10%) drug towards Enterobacteriaceae isolates which was approximately 31-50% more sensitive than other test drugs. Similar pattern was obtained for Non-enterobacteriaceae isolates (62.90%) where Elores contributed approximately 1-5% higher activity. However, Elores also conquered 79.51-86.59% resistance among meropenem, imipenem and piperacillin + tazobactam resistant pathogens.
 Conclusion: Susceptibility profile data revealed the equivalence of Elores (Antibiotic-adjuvant entity; AAE) with carbapenem drugs (meropenem and imipenem) and superiority over piperacillin + tazobactam against clinical pathogens. Elores was also found active towards meropenem, imipenem and piperacillin + tazobactam resistant pathogens. Therefore, Elores, a resistance breaker, can be used as an efficient treatment alternate towards infections caused by resistant pathogens.

Synergistic fungicidal activity with low doses of eugenol and amphotericin B against Candida albicans

Candida albicans frequently causes variety of superficial and invasive disseminated infections in HIV infected patients. Further, the emergence of non albicans species causing candidiasis predominantly in patients with advanced immune-suppression and drug resistance brings great apprehension. Hence, in this study we evaluate the capability of eugenol (EUG), a natural compound in combination with less toxic concentrations of amphotericin B (AmpB) for enhanced antifungal effects and reduced toxicity. Antifungal activity and time-kill assay were employed according to Clinical Laboratory Standard Institute (CLSI) guidelines with minor modifications on clinical isolates of Candida albicans. To confirm the synergistic interaction of EUG and AmpB, checkerboard experiments were employed. Interestingly, EUG-Amp B combination shows many fold higher anti-candida activity compared to single component treatment. Furthermore, our results depicts reactive oxygen species (ROS) driven killing and mitochondrial hyperpolarisation on treatment. Our data also suggests inhibition of calcium channel by EUG and predicts longer retainment of AmpB. Pronounced cellular damage was observed with combination treatment than to EUG and AmpB alone. Our finding is helpful for the removal of toxic concentrations of antifungal agents.

Prevalence and Genetic Relatedness of Extended Spectrum-β-Lactamase-Producing Escherichia coli Among Humans, Cattle, and Poultry in Pakistan.

Objective: To determine the prevalence and genetic relatedness of blaCTX-M-type extended spectrum-β-lactamase (ESBL)-producing Escherichia coli at the human-animal interface in Pakistan. Materials and Methods: A total of 150 human, cattle, and poultry fecal samples (50 each) were screened for ESBL-producing E. coli using ESBL CHROMagar®. Bacterial species confirmation as well as determination of minimum inhibitory concentrations (μg/mL) to different antibiotics was performed using the automated VITEK®-2 compact system. Phenotypic confirmation of ESBL production was performed according to the Clinical Laboratory Standards Institute (CLSI) guidelines. Genetic analysis of blaCTX-M was carried out by PCR and DNA sequencing. Plasmids and clonal similarity of the E. coli strains were determined by PCR-based replicon typing and pulsed-field gel electrophoresis (PFGE), respectively. Results: Of 150 samples, 29 (19.3%) ESBL-producing E. coli were recovered, and majority of them originated from human (n = 16; 55%), followed by cattle (n = 9; 31%) and poultry (n = 4; 13.7%). blaCTX-M-15 was predominant ESBL genotype (n = 25; 86.2%), mainly identified from human (n = 15) and cattle (n = 9). This is also the first report of the occurrence of CTX-M-15 and CTX-M-55 in cattle and poultry E. coli isolates of Pakistan, respectively. The majority of the ESBL-producing E. coli (96.5%) showed a multidrug resistance phenotype. All isolates carried IncFII or IncFIA plasmids, and the phylogroup B1 was dominant (44.8%) followed by phylogroups A (31%), D (17.2%), and B2 (6.8%). PFGE revealed that isolates from different hosts were genetically unrelated. Conclusion: Presence of CTX-M-15-type ESBL-producing E. coli in different reservoirs is alarming and has the potential to impact both veterinary and human therapeutic treatment options.

Resistance-related determinants in clinically relevant Staphylococcus aureus isolated from teaching therapeutic centers, Tehran, Iran

Objectives: The purpose of this study was to determine the antimicrobial resistance-related genes in Staphylococcus aureus isolated from patients referred to 14 teaching hospitals, Tehran, Iran. Methods: A total of 225 S. aureus were obtained from clinical samples in a period of 12 months. Antimicrobial resistance, minimum inhibitory concentration minimal inhibitory concentration and Inducible resistance phenotypes were determined based on the Clinical laboratory standard institute (CLSI) guidelines. PCR was performed for amplification of mecA/B/C, iles-2, ermA/B/C and ereA/B genes. Results: 39.5 and 40.8% of isolates were resistant to oxacillin and cefoxitin (FOX), respectively. The frequency of constitutive macrolide-lincosamide-streptograminB, inducible macrolide-lincosamide-streptograminB and MS phenotypes were 55.3, 28.6 and 16.1%, respectively. 40.8, 4, 7.5, 6.2, 16 and 3.1% of strains harbored the mecA, ileS-2, ermA, ermB, ermC and ereA genes, respectively. Conclusion: The frequency of methicillin-resistant S. aureus isolates in our hospitals was high and disk diffusion testing using FOX or oxacillin and/or FOX minimal inhibitory concentration E test as an alternative to PCR for identification of methicillin-resistant S. aureus is suggested. This study highlights the hypothesis that rapid testing plays an important role in antibiotic stewardship by getting patients on targeted therapy faster.

In vitro Susceptibility Pattern of Major Gram Negative Isolates to Selected Antimicrobial Agents

Background: The choice of choosing right anti-microbial therapy in hospitals depends on the knowledge of local anti-microbial susceptibility profile. This retrospective study was conducted to assess the in vitro susceptibility pattern of different pathogen isolates to various antibiotics including Cefepime-Amikacin-Antibiotic resistant breakers (ARBs)* in various hospitals across the Jaipur City.
 Methods: To characterize the antimicrobial susceptibility pattern of different isolates from various hospitals across the Jaipur City, a retrospective, observational analysis was done for antibiogram data. A total of 1201 Gram negative isolates collected during the period from January 2017 to December 2017 were included in the study. Antibiotic sensitivity testing was done in accordance with the recommendations of Clinical Laboratory Standard Institute (CLSI) guidelines.
 Results: Of the total 1201 Gram negative isolates included in this study, 51.6% were from wounds and pus specimens, 40.1% were from respiratory and 8.2% from blood. P. aeruginosa (49.7%) was the most frequently isolated pathogen distantly followed by A. baumannii (21.6%), K. pneumoniae (16.6%) and E. coli (12.1%). The highest susceptibility was reported to polymyxins (100%) including Colistin and Polymyxin B, among all the tested bacteria’s and system wise. Among all the antibiotic tested, (Cefepime-Amikacin-ARBs*) sensitivity ranged for 87.9% to 52% on pathogens (E. coli, K. pneumonia, P. aeruginosa) tested from samples of skin and soft tissue, respiratory tract, blood stream, followed by Meropenem ranged for 78.4% to 55% on pathogens (E. coli, K. pneumonia, P. aeruginosa), followed by ceftazidime-tazobactam ranged for 82.7% to 58% on pathogens (E. coli, K. pneumonia, P. aeruginosa) and 22.7% sensitive for A. baumannii to Cefoperazone sulbactam. Based on pathogen type, E. coli exhibited highest overall susceptibility and the lowest was reported by A. baumannii. The susceptibility of A. baumannii ranged from 1-26% to all the tested antibiotics except polymyxins with 100% susceptibility.
 Conclusions: This in vitro susceptibility data suggests that Cefepime-Amikacin-ARBs* can serve as important therapeutic option for the treatment of various resistant Gram-negative bacterial infections to relieve the excess pressure on last resort antibiotics, carbapenems and other drugs including Colistin and polymyxin B. Cefepime-Amikacin-ARBs*on the basis of antimicrobial susceptibility data can be considered as an effective therapeutic option for carbapenems in treating gram negative bacterial infections, and could be considered as a broad spectrum antibiotic sparer’s like carbapenem, colistin and Polymyxin B.

Prevalence of Extended Spectrum Beta-lactamase (ESBL) producing Enterobacteriaceae from clinical samples in a tertiary care hospital in Mumbai

Prevalence of Extended Spectrum Beta-lactamase (ESBL) producing Enterobacteriaceae from clinical samples in a tertiary care hospital in Mumbai - IJMR- Print ISSN No: - 2394-546X Online ISSN No:- 2394-5478 Article DOI No:- 10.18231/j.ijmr.2019.033, Indian Journal of Microbiology Research-Indian J Microbiol Res

Prevalence of Panton-Valentine Leukocidin (PVL) and Antimicrobial Resistance in Community-Acquired Clinical Staphylococcus aureus in an Urban Gambian Hospital: A 11-Year Period Retrospective Pilot Study.

Background: Staphylococcus aureus is a major human pathogen. Panton-Valentine leukocidin (PVL) is a virulence factor produced by some strains that causes leukocyte lysis and tissue necrosis. PVL-associated S. aureus (PVL-SA) predominantly causes skin and soft-tissue infections (SSTIs) but can also cause invasive infections such as necrotizing pneumonia. It is carried by both community-associated methicillin susceptible S. aureus (CA-MSSA) and methicillin resistant S. aureus (CA-MRSA). This study aims to determine the prevalence of PVL-SA among patients seen at an urban Gambian hospital and associated antibiotic resistance.Methods: Archived clinical S. aureus (70 invasive bacteraemia and 223 non-invasive SSTIs) from 293 patients were retrieved as well as relevant data from clinical records where available. Antibiotic susceptibility was assessed using disc diffusion according to Clinical Laboratory Standards Institute (CLSI) guidelines. Genomic DNA was extracted and the presence of lukF and lukS PVL genes was detected by conventional gel-based PCR.Result: PVL-SA strains accounted for 61.4% (180/293) of S. aureus isolates. PVL prevalence was high in both Gambian bacteraemia and SSTIs S. aureus strains. Antimicrobial resistance was low and included chloramphenicol (4.8%), cefoxitin (2.4%), ciprofloxacin (3.8%), erythromycin (8.9%), gentamicin (5.5%) penicillin (92.5%), tetracycline (41.0%), and sulfamethoxazole-trimethoprim (24.2%). There was no association of PVL with antimicrobial resistance.Conclusion: PVL expression is high among clinical S. aureus strains among Gambian patients. Reporting of PVL-SA clinical infections is necessary to enable the monitoring of the clinical impact of these strains in the population and guide prevention of the spread of virulent PVL-positive CA-MRSA strains.SUMMARY Staphylococcus aureus (S. aureus) is a major human pathogen with several virulence factors. We performed a retrospective analysis to investigate the prevalence of one such virulence factor (PVL) amongst clinical S. aureus samples. We found a high prevalence in our setting but antimicrobial resistance including methicillin resistance was low.

Microbiological profile and antibiotic susceptibility pattern of Gram-positive isolates at a tertiary care hospital.

OBJECTIVES:Gram-positive infections such as those by Staphylococcus aureus have contributed to the disease burden by increasing the morbidity and mortality rates in India. This study aims to determine the prevalence and the antibiotic susceptibility pattern of Gram-positive pathogens at a tertiary care hospital, Mumbai, Maharashtra, India.MATERIALS AND METHODS:This retrospective cross-sectional study was carried out from January, 2015 to December, 2017, at a tertiary care hospital in Mumbai, India. The clinical isolates were cultured, and identification was done using Vitek 2 culture system. The antibiotic susceptibility testing was done as per the Clinical Laboratory Standard Institute guidelines.RESULTS:Out of 2132 (29%) Gram-positive isolates, S. aureus (49%) was the most common encountered pathogen, followed by Enterococcus spp. (24.5%) and coagulase-negative Staphylococcus (16%). Majority of the S. aureus were observed in patients with skin and soft-tissue infections (61.2%) followed by those suffering from respiratory (41%) and bloodstream infections (35%). Among the infections caused by S. aureus, the prevalence of methicillin resistance was 30%. While the MRSA isolates showed lower sensitivity toward co-trimoxazole (39%), clindamycin (30%), erythromycin (23%), and ciprofloxacin (10%), they showed higher susceptibility to linezolid (98%), vancomycin (98%), and teicoplanin (98%). All the isolates were found to be sensitive to daptomycin and tigecycline. While vancomycin-resistant enterococci (VRE) formed 7.5%, the linezolid-resistant enterococcus species was as high as 4.1%.CONCLUSION:The study showed a high prevalence of MRSA and VRE, thereby emphasizing the increasing antimicrobial resistance pattern of the Gram-positive pathogens. Therefore, there is an urgent need for novel antimicrobial stewardship to restrict the ongoing resistance rate among the isolates.

Association between herd management practices and antimicrobial resistance in Salmonella spp. from cull dairy cattle in Central California.

BackgroundIn this study cull dairy cows from six California dairy herds were sampled seasonally over the course of a year. The objectives were to determine the prevalence of antimicrobial resistant (AMR) Salmonella spp. shed in cull cow feces, and the factors associated with fecal shedding of AMR and multidrug resistant (MDR) Salmonella.MethodsSix dairy farms located in the San Joaquin Valley of California were identified and enrolled as a convenience sample. On each dairy, and once during each of the four seasons, 10 cull cows were randomly selected for fecal sampling on the day of their removal from the herd. In addition, study personnel completed a survey based on responses of the herd manager to questions related to the previous 4 month’s herd management and the specific cattle sampled. Fecal samples were submitted to the California Animal Health and Food Safety laboratory for Salmonella isolation. Antimicrobial resistance was evaluated using broth microdilution method and a gram-negative assay plate following Clinical Laboratory Standards Institute (CLSI) guidelines and breakpoint references. All statistical models were survey adjusted for number of animals on sampling day.ResultsA total of 62 Salmonella were isolated from 60 of the 239 fecal samples collected. For 12% (95% confidence interval (CI) [3–20]) of fecal samples a multidrug resistant Salmonella was isolated. The survey-weighted results for the two most common drug classes for which isolates were resistant were tetracycline (39%; 95% CI [27–51]) and ampicillin (18%; 95% CI [9–27]). An important finding was the identification of cephalosporin as the third most common drug class for which isolates were resistant, with ceftriaxone (10%; 95% CI [2–17]) being the most common drug associated with resistance in that class. At the cow-level, reason for culling, prior treatment with antimicrobial drugs as the reason for culling was associated with higher odds of isolating an AMR Salmonella isolate. At the herd-level, percent of animals monthly culled on the farm as well as number of milking cows in the herd were associated with isolation of antimicrobial resistant Salmonella in cull cows.DiscussionSalmonella isolated from fecal samples from cull cows were resistant to important antimicrobials, such as ceftriaxone. The most common drug classes for which isolates were resistant were tetracyclines and beta-lactams, with ampicillin, ceftriaxone and ceftiofur being the three most common drugs within the latter. Cow and herd level factors were associated with isolating antimicrobial resistant Salmonella that should be further investigated for their potential role in promoting occurrence of AMR Salmonella. Our results also highlight the importance of monitoring dairy cattle sent to slaughter for shedding of Salmonella resistant to medically important antimicrobial drugs.