Genomic Surveillance of Clinical Pseudomonas aeruginosa Isolates Reveals an Additive Effect of Carbapenemase Production on Carbapenem Resistance.

Carbapenem resistance in Pseudomonas aeruginosa is increasing globally, and surveillance to define the mechanisms of such resistance in low- and middle-income countries is limited. This study establishes the genotypic mechanisms of ß-lactam resistance by whole-genome sequencing (WGS) in 142 P. aeruginosa clinical isolates recovered from three hospitals in Islamabad and Rawalpindi, Pakistan between 2016 and 2017. Isolates were subjected to antimicrobial susceptibility testing (AST) by Kirby-Bauer disk diffusion, and their genomes were assembled from Illumina sequencing data. ß-lactam resistance was high, with 46% of isolates resistant to piperacillin-tazobactam, 42% to cefepime, 48% to ceftolozane-tazobactam, and 65% to at least one carbapenem. Twenty-two percent of isolates were resistant to all ß-lactams tested. WGS revealed that carbapenem resistance was associated with the acquisition of metallo-ß-lactamases (MBLs) or extended-spectrum ß-lactamases (ESBLs) in the blaGES, blaVIM, and blaNDM families, and mutations in the porin gene oprD. These resistance determinants were found in globally distributed lineages, including ST235 and ST664, as well as multiple novel STs which have been described in a separate investigation. Analysis of AST results revealed that acquisition of MBLs/ESBLs on top of porin mutations had an additive effect on imipenem resistance, suggesting that there is a selective benefit for clinical isolates to encode multiple resistance determinants to the same drugs. The strong association of these resistance determinants with phylogenetic background displays the utility of WGS for monitoring carbapenem resistance in P. aeruginosa, while the presence of these determinants throughout the phylogenetic tree shows that knowledge of the local epidemiology is crucial for guiding potential treatment of multidrug-resistant P. aeruginosa infections. IMPORTANCE Pseudomonas aeruginosa is associated with serious infections, and treatment can be challenging. Because of this, carbapenems and ß-lactam/ß-lactamase inhibitor combinations have become critical tools in treating multidrug-resistant (MDR) P. aeruginosa infections, but increasing resistance threatens their efficacy. Here, we used WGS to study the genotypic and phylogenomic patterns of 142 P. aeruginosa isolates from the Potohar region of Pakistan. We sequenced both MDR and antimicrobial susceptible isolates and found that while genotypic and phenotypic patterns of antibiotic resistance correlated with phylogenomic background, populations of MDR P. aeruginosa were found in all major phylogroups. We also found that isolates possessing multiple resistance mechanisms had significantly higher levels of imipenem resistance compared to the isolates with a single resistance mechanism. This study demonstrates the utility of WGS for monitoring patterns of antibiotic resistance in P. aeruginosa and potentially guiding treatment choices based on the local spread of ß-lactamase genes.

Prevalence of latent tuberculosis infection in healthcare workers in tertiary care hospitals of Pakistan.

OBJECTIVE: To determine the prevalence of latent tuberculosis infection (LTBI) in healthcare workers in tertiary care hospitals of Rawalpindi, using interferon gamma release assay. METHODS: It was a cross-sectional study. The samples were collected from pulmonology and microbiology departments of three hospitals; i) Military Hospital, Rawalpindi, ii) Fauji Foundation Hospital, Rawalpindi and iii) Pakistan Institute of Medical Sciences, Islamabad. The study was completed in one year from January 2017 to January 2018. Fifty-five asymptomatic healthcare workers of both genders between the ages of 18-50 years with a working tenure of at least one year in concerned departments were included and those with active tuberculosis were excluded from the study. Whole blood from subjects was collected and plasma was checked for interferon gamma value by IGRA (Interferon gamma release assay). RESULTS: In this study of total 55 healthcare workers a high prevalence 22 (40.0%) of latent tuberculosis was found. When LTBI distribution was analyzed within occupational categories, the most frequently affected were sanitary workers 3 (100.0%), nurses 5 (50.0%), doctors 6 (43%) and nursing assistants 2 (40%). CONCLUSION: The prevalence of LTBI in healthcare workers is alarmingly high in our local healthcare settings.

Phenotypic Identification, Frequency Distribution and Antibiogram of Carbapenemase Producing Enterobacteriaceae in Clinical Isolates.

OBJECTIVE: To differentiate between Ambler class A, B and D of carbapenemase-producing Enterobacteriaceae by using simple phenotypic methods that can be carried out in the laboratory without requiring any specialised techniques. STUDY DESIGN: Cross-sectional study. PLACE AND DURATION OF STUDY: Microbiology Department, Army Medical College, NUST, Islamabad, from November 2015 to November 2016. METHODOLOGY: Clinical specimens were subjected to identification of Enterobacteriaceae by colony morphology and API 20 E. Carbapenem resistance was detected by applying meropenem disc (10 µg) by disc diffusion method according to CLSI (Clinical Laboratory Standard Institute) criteria. Carbapenemase production among Enterobacteriaceae was detected by Modified Hodge test. Phenotypic methods, Phenylboronic acid (for Class A KPC producing Enterobacteriaceae) and EDTA inhibition tests (for Class B MBL producing Entrobacteriaceae) were applied. Presence of OXA 48 was detected by phenotypic method using imipenem 10 µg, EDTA and PBA discs. Antibiotic susceptibility was determined by Kirby-Bauer disc diffusion method. RESULTS: Forty-three out of 45 (95.45%) were carbapenemase producers. Thirty-eight out of 43 (88.3%) were KPC producers and 4 out of 43 (11.62%) were MBL producers. All KPC producers were Klebsiella pneumoniae. Among five MBL producers, one each (20%) was Enterobacter cloacae and Escherichia coli and 3 (60%) were Klebsiella pneumoniae. All MBL producers were resistant to aztreonam and amoxicillin/clavulanate. Two of the KPC producing Klebsiella pneumoniae were pan-drug resistant (resistant to colistin and tigecycline). Two were non-carbapenemase producers. CONCLUSION: Enterobacteriaceae strains producing KPC-type carbapenemase were the most prevalent (88.3%) in the studied healthcare setup.

Draft Genome Sequence of a Salmonella enterica Serovar Typhi Strain Resistant to Fourth-Generation Cephalosporin and Fluoroquinolone Antibiotics.

Typhoid is endemic in developing countries. We report here the first draft genome sequence of a Salmonella enterica serovar Typhi clinical isolate from Pakistan exhibiting resistance to cefepime (a fourth-generation cephalosporin) and fluoroquinolone antibiotics, two of the last-generation therapies against this pathogen. The genome is ~4.8 Mb, with two putative plasmids.

Extended Spectrum Beta Lactamase producing Cephalosporin resistant Salmonella Typhi, reported from Rawalpindi, Pakistan.

Typhoid is endemic in many parts of southeast Asia. Due to the resistance of the organism to first line of antibiotics (ampicillin, chloramphenicol, cotrimoxazole) as well as to fluoroquinolones, third generation cephalosporins have been in use for the empiric treatment of typhoid for years. However an increasing incidence of Salmonella Typhi is being reported sporadically from various regions. We report a case of typhoid due to Salmonella Typhi which was non-responsive to treatment with a cephalosporin, was found to be multidrug resistant and resistant to ciprofloxacin and third generation cephalosporin as well. The patient was finally treated successfully with intravenous administration of a carbapenem.

ICU Pathogens: A Continuous Challenge.

OBJECTIVE: To determine the frequency and antibiogram of pathogens in an intensive care unit (ICU). STUDY DESIGN: Cross-sectional, observational study. PLACE AND DURATION OF STUDY: Department of Microbiology, Army Medical College, National University of Science and Technology, Islamabad, from January 2013 to January 2014. METHODOLOGY: Clinical samples, received from patients admitted in ICU, were inoculated on various medias like blood agar, chocolate agar, MacConkey agar and urine samples on CLED. These were then incubated at 37°C for 24 hours. Isolates were identified by colony morphology, Gram reaction, catalase test, oxidase test. Species identification in case of Gram Negative Rods was done by using API 20E (BioMérieux). Antibiotic susceptibility was done by using modified KirbyBauer disc diffusion technique. Bacterial isolates were prepared and inoculated on Mueller-Hinton agar plates followed by application of various antibiotic disc (Oxoid, UK) as per manufacturer's instructions. The plates were then incubated at 37°C aerobically for 18 - 24 hours. Zone diameters were measured and interpreted as sensitive and resistant, according to Clinical and Laboratory Standards Institute (CLSI) guidelines. RESULTS: Out of the 367 positive cultures, 116 (31.08%) were Acinetobacter baumanniisusceptible to minocycline and tigecycline followed by Klebsiella pneumoniae (n=71, 16%) susceptible to tigecycline and meropenem. Others were Pseudomonas aeruginosa, Escherichia coli,Coagulase Negative Staphylococcus, Staphylococcus aureus, Enterococcus spp., Streptococcus spp., Klebsiella oxytoca, Stenotrophomonas maltophilia,and Candida spp. CONCLUSION: Acinetobacter baumanniiwas the most frequently isolated pathogen. Most of the cultures yielding pathogens were from respiratory tract samples. Gram negative isolates were multidrug resistant but most were tigecycline and susceptible to meropenem.

Methicillin resistant coagulase negative staphylococcus: From colonizer to a pathogen.

The objective of our study was to determine the frequency of methicillin resistance in coagulase negative Staphylococcus (CoNS) and to determine its in-vitro antimicrobial susceptibility to various other routinely used antibiotics. It was a cross sectional study conducted at the department of Microbiology, Army Medical College, Rawalpindi, Pakistan from June 2011 to May 2012. The organisms were identified on the basis of colony morphology, Gram staining, catalase, DNAase and slide/tube coagulase tests. The organisms were considered to be methicillin resistant when the diameter of zone of inhibition was less than 25mm around 30µg cefoxitin disc. Antibiotic sensitivity was determined using the Modified Kirby-Bauer disc diffusion method. From a total of 337 CoNS, 201 were methicillin resistant and were included in the study. All were resistant to Penicillin, followed by Erythromycin (93•1%), Ciprofloxacin (77%), Co-trimoxazole (74•8%), Gentamicin (68•3%), Clindamycin (51•06%), Tetracycline (44•6%), Fusidic acid (40%), Rifampicin (39•5%), Chloramphenicol (19•3%), Linezolid (2%), Minocycline (1•1%), and Vancomycin (0%). More than half of CoNS were methicillin resistant. Vancomycin is the only drug to which all of the MRCoNS were sensitive, with more than 98% of the isolates being sensitive to Linezolid and Minocycline.

Evaluation of phenotypic tests for detection of Amp C beta-lactamases in clinical isolates from a tertiary care hospital of Rawalpindi, Pakistan.

OBJECTIVE: To evaluate sensitivity and specificity of disc approximation test compared to three-dimensional extract test as a phenotypic gold standard test for detection of AmpC beta-lactamase producing Escherichia coli and Klebsiella pneumoniae. METHODS: The cross-sectional validation study was conducted from November 2014 to April 2015 at Army Medical College, Rawalpindi, Pakistan. Extended spectrum beta lactamases (ESBLs) were isolated from various clinical specimens. Screening for AmpC beta-lactamases was done by using cefoxitin disc (30µg) showing inhibition zone diameter of <18mm. Screen-positive isolates were subjected to disc approximation test (DAT) and three-dimensional extract test(3-DET).SPSS 20 was used for statistical analysis. RESULTS: A total of 120 ESBL producing Gram negative rods were included in the study. Out of these 120, 82(68.33%) were found to be AmpC beta-lactamase producing on screening with cefoxitin disc. Amongst these 82 isolates, Escherichia coli were identified in 57(69.51%) and Klebsiella pneumoniae in 25 (30.48%). Phenotypic confirmation by disc approximation test (DAT) identified 43(52.43%). AmpC beta-lactamase producing isolates, whereas gold standard 3-DET showed 38(46.34%) of AmpC beta-lactamase producing isolates. Hence, sensitivity of disc approximation test (DAT) was found to be 88%, specificity was 92%, positive predictive value was 92.68%, negative predictive value was 87.80% and diagnostic accuracy was 90.24%. CONCLUSIONS: Implementation of disc approximation test in the laboratories can help in identifying AmpC beta-lactamase harbouring organisms.

In Vitro Efficacy of Doripenem against Pseudomonas aeruginosa and Acinetobacter baumannii by E-Test.

OBJECTIVE: To assess the in vitro efficacy of doripenem against Pseudomonas aeruginosa and Acinetobacter baumannii using Epsilometer strips. STUDY DESIGN: Cross-sectional study. PLACE AND DURATION OF STUDY: Department of Microbiology, Army Medical College, Rawalpindi and National University of Sciences and Technology, Islamabad, from May 2014 to September 2014. METHODOLOGY: A total of 60 isolates of Acinetobacter baumannii and Pseudomonas aeruginosa collected from various clinical samples received from Military Hospital were included in the study. The specimens were inoculated onto blood, MacConkey and chocolate agars. The isolates were identified using Gram staining, motility, catalase test, oxidase test and API 20NE (Biomeriux, France). Organisms identified as Acinetobacter baumannii and Pseudomonas aeruginosa were included in the study. Bacterial suspensions equivalent to 0.5 McFarland turbidity standard of the isolates were prepared and applied on Mueller Hinton agar. Epsilometer strip was placed in the center of the plate and incubated for 18-24 hours. Minimum Inhibitory Concentration (MIC) was taken to be the point where the epsilon intersected the E-strip. MIC of all the isolates was noted. RESULTS: For Pseudomonas aeruginosa isolates, MIC(50) was 12 µg/mL and MIC(90) was 32 µg/mL. For Acinetobacter baumannii MIC(50) and MIC(90) was 32 µg/mL. CONCLUSION: Doripenem is no more effective against Pseudomonas aeruginosa and Acinetobacter baumannii in our setting.

Early Diagnosis of Typhoid By PCR For FliC-d Gene of Salmonella Typhi in Patients Taking Antibiotics.

OBJECTIVE: To compare PCR (Polymerase Chain Reaction) with blood culture, typhi-dot and Widal test for the diagnosis of typhoid in patients taking antibiotics. STUDY DESIGN: Cross-sectional, comparative study. PLACE AND DURATION OF STUDY: National University of Sciences and Technology, Islamabad, Pakistan, from April 2013 to August 2014. METHODOLOGY: One hundred and five patients were included in the study. Blood was collected and inoculated into tryptone soya broth for culture. Any growth obtained was identified by API 20 E and confirmed by Salmonella anti-sera. Typhi-dot and Widal test were also done on all the samples. DNA extraction was done and PCR was carried out. RESULTS: Among the 105 patients, 79 (75.2%) were males and 26 (24.8%) were females, with mean age of 20.64 ±14 years. Typhi-dot was positive in 58 (55.2%) and negative in 47 (44.8%) patients. Blood widal test was positive in 27 (25.7%) and negative in 78 (74.3%) patients. Salmonella Typhi was positive on blood culture in only one (1%) patient. PCR for Salmonella Typhi was positive in 102 (97.1%) and negative in 3 (2.9%) patients. Positive cases detected by PCR were significantly higher as compared to Typhi-dot (p < 0.001), blood Widal test (p < 0.001) and blood culture (p < 0.001). CONCLUSION: Positivity rate of PCR was significantly higher as compared to blood culture, Typhi-dot or Widal test for diagnosing typhoid in patients who were already taking antibiotics.

Coagulase negative staphylococci - a fast emerging threat.

OBJECTIVE: To determine the frequency of isolation of coagulase-negative staphylococci and their resistance to methicillin over a period of time. METHODS: The descriptive cross-sectional study was carried out at Army Medical College, Rawalpindi, from June 2009 to May 2012, and comprised clinical samples mostly from patients admitted to the intensive care unit. They were inoculated onto appropriate culture media depending upon the specimen. After 24-hour incubation at 35°C, coagulase-negative staphylococci were identified on the basis of colony morphology, gram staining, a positive catalase and a negative tube coagulase test.Methicillin resistance among the isolated staphylococci was determined using a 30µg Cefoxitin disc as per the Clinical and Laboratory Standards Institute protocol. Number of coagulase-negative staphylococci for each year and their methicillin resistance rates were calculated. A comparison was made with methicillin resistant staphylococcus aureus) isolated during the same period. RESULTS: Of the total 1331 specimens studies over three years, 581(43.65%) were coagulase-negative staphylococci. The rate of coagulase-negative staphylococci and methicillin resistance was higher each year; 110(26.6%) in May 2009-Jun 2010, 134(36.5%) in 2011, and 337(61%) in 2012. Methicillin resistance rates also increased from 25(22.7%) to 46(34.3%) and then to 201(59.6%) in 2012.Maximum isolated specimens came from blood 311(53.5%), followed by pus/swabs 204(35.1%). CONCLUSIONS: The frequency of isolation of coagulase-negative staphylococci and its methicillin resistance among hospitalised patients is on the rise.

Comparative Efficacy of Ceftaroline with Linezolid against Staphylococcus aureus and Methicillin Resistant Staphylococcus aureus.

OBJECTIVE: To compare the in vitro antimicrobial efficacy of ceftaroline with linezolid against Staphylococcus aureus and methicillin resistant Staphylococcus aureus. STUDY DESIGN: Quasi-experimental study. PLACE AND DURATION OF STUDY: Microbiology Department, Army Medical College, Rawalpindi, from January to December 2013. METHODOLOGY: Clinical samples from respiratory tract, blood, pus and various catheter tips routinely received in the Department of Microbiology, Army Medical College, Rawalpindi were innoculated on blood and MacConkey agar. Staphylococcus aureus was identified by colony morphology, Gram reaction, catalase test and coagulase test. Methicillin resistant Staphylococcus aureus detection was done by modified Kirby Bauer disc diffusion method using cefoxitin disc (30 µg) and the isolates were considered methicillin resistant if the zone of inhibition around cefoxitin disc was ≤ 21 mm. Bacterial suspensions of 56 Staphylococcus aureus isolates and 50 MRSA isolates were prepared, which were standardized equal to 0.5 McFarland's turbidity standard and inoculated on Mueller-Hinton agar plates followed by application of ceftaroline and linezolid disc (Oxoid, UK), according to manufacturer's instructions. The plates were then incubated at 37 °C aerobically for 18 - 24 hours. Diameters of inhibition zone were measured and interpretated as per Clinical and Laboratory Standards Institute (CLSI) guidelines. RESULTS: Out of 106 isolates all of the 56 Staphylococcus aureus (100%) were sensitive to ceftaroline and linezolid. However, out of 50 methicillin resistant Staphylococcus aureus, 48 (96%) were sensitive to ceftaroline whereas, 49 (98%) were sensitive to linezolid. CONCLUSION: Ceftaroline is equally effective as linezolid against Staphylococcus aureus and methicillin resistant Staphylococcus aureus.

Comparison of urine dipstick test with conventional urine culture in diagnosis of urinary tract infection.

OBJECTIVE: To determine the sensitivity, specificity, Positive Predictive Value (PPV) and Negative Predictive Value (NPV) of Urine Nitrite (NIT) and Leukocyte Esterase (LE) test compared with urine culture for diagnosis of UTI. STUDY DESIGN: Validation study. PLACE AND DURATION OF STUDY: Department of Microbiology, Army Medical College, Rawalpindi, from January 2013 to December 2013. METHODOLOGY: Three hundred fresh uncentrifuged urine samples with suspicion of UTI, were collected and tested for LE and NIT by using (COMBI-10SL, UK) strip. Nitrite was considered as positive if there was a change in color of dipstick from colorless towards pink within 60 seconds. Leukocyte esterase was considered as positive if there was a change in color from off-white towards purple within 2 minutes. Quantitative urine culture was performed by using the strips calibrated to deliver 0.02 ul of urine on Cystine Lactose Electrolyte Deficient (CLED) medium agar. All plates were incubated at 37°C and read after 24 and 48 hours. Culture was considered as gold standard to evaluate the performance of dipstick test. RESULTS: Out of 300 samples, 136 were culture positive and 164 were culture negative. Out of 136 positive culture results, 103 were dipstick positive and 33 were negative. Sensitivity, specificity, positive predictive value and negative predictive value of both nitrite and leukocyte esterase were 75.74%, 68.90%, 66.66% and 77.40% respectively considering culture as gold standard. CONCLUSION: Dipstick test for the detection of leukocyte esterase and nitrite in urine are sensitive and specific and can be used reliably for the detection of UTI in resource limited setup.

Frequency and antibiogram of vancomycin resistant enterococcus in a tertiary care hospital.

OBJECTIVE: To determine the frequency of Vancomycin Resistant Enterococcus (VRE) in a tertiary care hospital of Rawalpindi, Pakistan. STUDY DESIGN: Observational, cross-sectional study. PLACE AND DURATION OF STUDY: Department of Microbiology, Army Medical College, Rawalpindi, from May 2011 to May 2012. METHODOLOGY: Vancomycin resistant Enterococcus isolated from the clinical specimens including blood, pus, double lumen tip, ascitic fluid, tracheal aspirate, non-directed bronchial lavage (NBL), cerebrospinal fluid (CSF), high vaginal swab (HVS) and catheter tips were cultured on blood agar and MacConkey agar, while the urine samples were grown on cystine lactose electrolyte deficient agar. Later the antimicrobial susceptibility testing of the isolates was carried out using the modified Kirby-Bauer disc diffusion method on Mueller Hinton agar. RESULTS: A total of 190 enterococci were isolated. Of these, 22 (11.57%) were found to be resistant to vancomycin. The antimicrobial sensitivity pattern revealed maximum resistance against ampicillin (86.36%) followed by erythromycin (81.81%) and gentamicin (68.18%) while all the isolates were 100% susceptible to chloramphenicol and linezolid. CONCLUSION: The frequency of VRE was 11.57% with the highest susceptibility to linezolid and chloramphenicol.

Association between periurethral colonization with uropathogens and subsequent bacteriuria in catheterized patients.

OBJECTIVE: To determine the number of catheterized patients who develop bacteriuria due to the presence of organisms in their periurethral flora, which may subsequently cause Urinary Tract Infection (UTI) in these patients. STUDY DESIGN: Non-interventional, cohort study. PLACE AND DURATION OF STUDY: This study was conducted on patients of Medical Intensive Care, Surgical and Urology Units of Combined Military Hospital, Lahore, from February to April 2006. METHODOLOGY: A total of 60 hospitalized patients, who were catheterized for various underlying diseases, were included in the study. Urine samples and periurethral swabs were obtained from all patients and cultured on appropriate culture media. Various tests used for the identification of microorganisms were: Gram-staining, catalase test, coagulase test and esculin hydrolysis for the identification of Gram-positive bacteria, API 20e for Gram-negative bacilli, whereas lactophenol blue preparation and germ tube test were used for the identification of yeasts. RESULTS: Out of 60 patients, 41(68.3%) were males and 19 (31.7%) were females. The mean duration of catheterization was 4.5 days. In males, culture of periurethral swabs revealed coagulase negative staphylococci in 11 (40.7%), Staphylococcus aureus in 10 (37%) and Enterococcus fecalis in 3 (11.1%) patients. In females, the organisms isolated were coagulase negative staphylococci in 4 (25%), Staphylococcus aureus in 4 (25%), Enterococcus fecalis in 4 (25%), Pseudomonas aeruginosa in 2 (12.5%), Escherichia coli in 3 (18.6%) and Candida albicans in 3 (18.6%) patients. Twenty nine patients developed bacteriuria (p < 0.05). Escherichia coli was the commonest organism causing bacteriuria in either gender followed by other Gram-negative organisms. Coagulase negative Staphylococcus was isolated in the urine of one male patient only. In males, 2 (10%) out of 20 patients with Gram-negative bacteriuria were colonized by the same organism, whereas in females, 4 (44.4%) out of 9 bacteriuric patients were colonized by the same organism. CONCLUSION: Predominantly Gram-positive organisms colonized the periurethral area in males as well as in the majority of females, whereas Gram-negative bacteria were mainly responsible for the bacteriuria in both genders. There was a significant association between periurethral colonization and subsequent bacteriuria, however, prior colonization with a particular organism is not a decisive event in the initiation of bacteriuria.