Standard Microbiological Tests Research Articles

Investigation of Pseudomonas aeruginosa bacteria in a number of Baghdad schools and extent of their resistance to disinfectants and sterilizers

PurposeThis study was design to investigate of P. aeruginosa, an example of Gram-negative bacteria, in seven primary and secondary schools of Baghdad city, and the effects of Ethanol and Dettol of P. aeruginosa biofilm.Design/methodology/approachSeventy swabs were collected from seven primary and secondary schools of Baghdad city, Iraq, during November -December 2022. Swabs were collected from classes desk, doors handles, students hands and water taps. Standard microbiological testing methods were used on the samples for isolation and identification. The ability of bacteria to form biofilm and the effects of Ethanol and Dettol on"preformed” biofilms was examined by microtiter plate with the use of an ELISA reader.FindingsIn 70 swabs from seven primary and secondary schools, growth was observed in 33 swabs as P. aeruginosa. Primary schools were higher contaminated than secondary and water taps and door handles represented the main source of this contamination. The ability of bacteria to produce biofilm was observed in 19 (57.6%) isolates and 14 (42.4%) nonbiofilm producers. As well as, Ethanol (70%) treatment of preformed biofilms led to enhance biofilm formation and revealed significantly greater staining after 4 and 24h than Dettol (3%) compared to an untreated control (tryptic soy broth (TSB) incubation).Originality/valueStudies on P. aeruginosa in Iraqi schools are quite rare. This work is considered distinctive because it drew attention to the presence of pathogenic bacteria within primary and secondary schools, which are not considered their natural environment.

Capsular gene distribution and RAPD typing of Streptococcus agalactiae isolated from pregnant women

Streptococcus agalactiae has different virulence factors, from which the capsule has the most significant role in the pathogenesis of this organism. We aimed to investigate the distribution of more prevalent capsular genes among different Random Amplified Polymorphic DNA (RAPD) types of S. agalactiae isolated from pregnant women. A total of 106 isolates were collected from 420 vaginal and rectal swabs obtained from pregnant women. The specimens were transferred using Todd Hewitt Broth and were cultured on a blood agar containing antibiotics. The S. agalactiae isolates were identified by the standard microbiological and biochemical tests. The genomic DNAs of S. agalactiae isolates were extracted using an extraction kit. Then, the PCR method was used to detection of the capsular genes. Moreover, The RAPD PCR was used to genotyping of the isolates. The colonization rate of the pregnant women was 25.23%, and there was a statistically significant correlation between the weeks of gestation and the probability of colonization (p-value < 0.05). Also, 31 (29.24%) and 18 (16.98%) pregnant women had a history of abortion and membrane rupture, respectively. In addition, 20 (18.86%), 32 (30.18%), 4 (3.77%), and 6 (5.66%) isolates carried genes encoding capsular types Ia, Ib, III, and V, respectively. None isolates had the type II capsular gene, and other 44 isolates were non-typeable. Nine clones (clusters) of S. agalactiae were observed in the present study with 70% similarity, and 53 different types were identified among the isolates. Except for capsular types III and V that belonged to clones 3, 5, 7, and 9, other capsular types were detected in different RAPD types. We found that the capsular types Ib and Ia were predominant among pregnant women in this area, indicating their significance for vaccine designation. Also, our isolates showed a lower genotypic diversity in RAPD typing. This may be due to the same sources of most isolates.

Prospective cohort study of surgical site infections following single dose antibiotic prophylaxis in caesarean section at a tertiary care teaching hospital in Medchal, India.

Caesarean section (CS) is considered to be a life-saving operative intervention for women and new-borns in certain antepartum and intrapartum conditions. Caesarean delivery may be accompanied by several complications including surgical site infections (SSI). However, there is a significant lack of uniformity in the administration of antibiotics for preventing surgical site infections (SSI) following caesarean deliveries. The present study was conducted to determine the incidence of post CS SSI following the adoption of single-dose antibiotic prophylaxis as recommended by WHO at a tertiary care teaching hospital in Medchal, India. Also, to identify the risk factors of SSI and reported the bacteriological profiles and the antimicrobial susceptibility pattern of the culture positive isolates. To estimate the incidence of surgical site infections (SSI's) according to CDC criteria following WHO-recommended single-dose antibiotic prophylaxis for caesarean section at a tertiary care teaching hospital in Medchal, India. A prospective hospital-based study was conducted between June 2017 and December 2019, in which women who underwent caesarean delivery were followed up for 30 days post-delivery. Clinical details were collected using a structured questionnaire, and participants were followed up weekly after discharge to document any signs and symptoms of SSI. Symptomatic patients were requested to come to the hospital for further investigation and treatment. Standard microbiological tests were conducted to detect microorganisms and their antibiotic sensitivity. The study included 2,015 participants with a mean age of 24.1 years. The majority were multigravida (n = 1,274, 63.2%) and underwent emergency caesarean delivery (n = 1,232, 61.1%). Ninety two participants (4.6%, 95% CI: 3.7% to 5.6%) developed surgical site infections, with 91 (98.9%) having superficial and 1 (1.1%) having a deep infection. Among those who developed an SSI, 84 (91.3%) did so during their hospital stay, while 8 (8.7%) developed an SSI at home. The adjusted relative risk (a RR) for developing an SSI was 2.5 (95% CI: 1.4 to 4.6; power 99.9%) among obese women and 2.3 (95% CI: 1.1 to 4.7; power 100%) among women aged 25 years or younger. Microbial growth in culture was observed from 55 (75.8%) out of total 66 samples. The most common organisms identified were Staphylococcus aureus (n = 7(12.3%)23, 46.0%), Klebsiella sp. (n = 13, 26.0%), and Escherichia coli (n = 12, 24.0%). The rate of SSI following caesarean deliveries subjected to single dose antibiotic prophylaxis was low. Young women and obese women were at high risk of developing SSI.

Physician-directed microbiological testing versus syndromic multiplex PCR in gastroenteritis.

Syndromic multiplex PCR testing is an alternative to conventional stool testing based on physician-directed request forms. The objective of this study was to compare the etiologic yield of conventional microbiological testing based on physician-directed request forms with that of rapid syndromic testing. In addition, the adequacy of the clinician ordering, which is an important piece of the diagnostic stewardship, was evaluated. Physician-directed conventional microbiological testing and extensive molecular syndromic testing with the Fast Track Diagnostics Gastroenteritis Kit were performed in parallel on 1238 samples to evaluate the contribution of a multiplex panel to the diagnostic process of gastroenteritis. A potential causative pathogen was identified in 18.4% of stool samples by standard microbiological testing and in 41.3% of stool samples tested using the syndromic panel. Only 15.1% of the request forms could be considered successful of which 88.2% were labeled inadequate. Conventional physician-directed based testing missed the etiologic diagnosis in 32.3% of the specimens (excluding sapovirus and astrovirus). Bacterial infections were theoretically not missed as bacterial stool culture was requested on all stool samples, but in 28.6% of the cases, no isolate could be recovered. In 36.9% of the samples testing positive for a viral pathogen, no viral testing was requested. In addition, 72.5% of all samples positive for a parasite were clinically suspected by the physician. This study suggests that syndromic multiplex PCR assays are a better strategy for pathogen detection in patients with gastroenteritis than physician-directed laboratory testing based on the clinical presentation.

Phenotypic and Genetic Characterization of Polyhydroxyalkanoate Producing Bacterial Isolates from Hypersaline Water Body, Atlantic Ocean, Nigeria

Introduction: The importance of polyhydroxylalkanoate (PHA) producing bacteria is hinged on the basis that PHA itself possess environmentally advantages over synthetic plastics therefore, the proper identity of these bacteria is inevitable.&#x0D; Aim: To determine the bacteria identification methods of phenotypic and genetic identification for marine sourced effective polyhydroxylalkanoate (PHA) production.&#x0D; Study Design: Randomised design was employed for PHA producing bacterial isolates.&#x0D; Place and Duration of Study: Samples containing PHA-producing bacteria from the hypersaline water body of Nigerian Southern Atlantic Ocean (6.35ᴼ N 3.28ᴼ E -St1; 6.35ᴼ N 3.40ᴼ E -St2; and 6.36ᴼ N 3.47ᴼ E -St3); water depths (0.07m (top), 50m (middle) and 100m (bottom) were collected and investigated between August, 2016-September, 2019.&#x0D; Methodology: Bacterial isolates was characterized using standard microbiological and biochemical tests for the phenotypes, and 16S rDNA for the genotypes.  Isolates were also screened for PHA potentials using specific primers.&#x0D; Results: Genetically identified Bacillus cereus strains LB17 and Bacillus paramycoides SA27 were phenotypically identified as Bacillus badius. Similarly, Acinetobacter calcoaceticus JL11, five strains of Alcaligenes faecalis, seven strains of Bacillus spp., Enterobacter cloacae, Falsochrobactrum ovis, Ochrobactrum ciceri, Providencia stuartii including two species of Pseudomonas and Bordetella trematum were all phenotypically identified as Corynebacterium kutsceri. Invariably, Alcaligenes faecalis (strains PSD10 and DEP8), Pseudomonas aeruginosa (H47921) and Vagococcus fluvialis (AWW1) were identified as Staphylococcus species. All 34 bacterial isolates expressed phaC genes for PHA potential, while Alcaligenes faecalis strains and Bacillus humi (NBPP9) expressed phaC1 for short PHA chain.&#x0D; Conclusion: This study established variations in the genetical identity of Corynebacterium kutsceri, Enterobacter amnigenus, Micrococcus luteus, Micrococcus varians, Staphylococcus epidermidis and Staphylococcus saprophyticus when compared with the phenotypic identification methods. But, there was synchronization in the genotypic and phenotypic identity of bacillus species. Records of varietal differences of PHA potential were also obtained from same species in this research. It is therefore pertinent to rely on the genetic identification of bacteria for the effective determination of PHA-producing character as displayed in Bacillus spp.

Extended-Spectrum Beta-Lactamases Genes in Klebsiella pneumoniae Isolates Obtained from Patients in Intensive Care Units

Background: Drug-resistant hospital-acquired infections (HAIs) are a growing concern in modern medicine throughout the world. Klebsiella pneumoniae is one of the most prominent causative agents of multidrug-resistant nosocomial infections. It is also widely recognized for having a high resistance level to many antibiotic classes, particularly beta-lactams. Carbapenemase-producing K. pneumoniae has been identified as a major global cause of HAIs with adverse clinical outcomes. Therefore, it is of the utmost importance to have an in-depth understanding of the antimicrobial resistance (AMR) genetic determinants of this bacterium to stop the spread of highly resistant K. pneumoniae in healthcare facilities and the resulting patient morbidity and mortality. Objectives: This study aimed to investigate the AMR pattern of K. pneumoniae isolates obtained from intensive care units (ICUs), with a focus on extended-spectrum beta-lactamases (ESBLs) genes blaCTX-M, blaGES, and blaIMP. Methods: A total of 105 K. pneumoniae isolates obtained from the sputum samples of ICU patients were identified and confirmed using standard microbiological tests and 16S rRNA polymerase chain reaction (PCR). The antibiotic susceptibility test was performed for all the isolates. The presence of ESBL genes was determined phenotypically and by PCR. Results: The highest level of resistance was observed against ceftazidime (100%), cefotaxime (99%), and imipenem (93.3%). Approximately 87.6% and 39% of the isolates were sensitive to colistin and gentamicin, respectively. Phenotypic ESBL production was observed in 16 isolates, and the prevalence of blaCTX-M was 86.7%. No blaGES and blaIMP genes were detected. Conclusions: Periodic investigation of AMR-mediating genes is essential due to the high prevalence of ESBL genes in HAIs. The presence of other ESBL genes needs to be investigated for a more accurate understanding of the AMR status of K. pneumoniae in healthcare settings.

Isolation and characterization of bacteria from babies heat prickly powder purchased in Umuahia, Abia state

Aim: To isolate and Characterize Bacteria from Babies Heat Prickly powders purchased in Umuahia, Abia state Method: A total of Ten (10) Baby heat prickly powder samples were randomly purchased from different market locations in Umuahia, Abia state. Isolation and identification of Bacteria were conducted using standard microbiological techniques. Bacteria were Characterized using standard microbiological and biochemical test. Results: The isolated Bacteria include Staphylococcus aureus, Bacillus sp, Staphylococcus epidermidis and Pseudomonas aeruginosa. Staphylococcus aureus had the highest percentage occurrence (40%) while other Bacteria had (20%) each with the total bacteria count (TBC) ranging from 4.3 ×104 CFU/g to 1.32 ×105 CFU/g. Conclusion: High bacterial load was observed in the present study which showed there was contamination of most of the products in respect to the international Microbiological standards recommended limits for bacterial contamination in powder/Cosmetic products (1.0 x 103 CFU/g) for bacteria. This could be as a result of poor manufacturing practices, poor hygiene, contaminated raw materials or the susceptibility of the ingredients contained in the baby powders.

Prevalence study of methicillin resistant &lt;i&gt;Staphylococcus aureus&lt;/i&gt; and its SCC&lt;i&gt;mec&lt;/i&gt; features in horses and handlers in Zaria and Kaduna, Nigeria

Methicillin resistant Staphylococcus aureus (MRSA) is a pathogen with public health implications being resistant to most used antibiotics. It has been associated with health facilities but has now become prevalent in community settings. The presence of MRSA in 240 apparently healthy horses and 65 horse handlers in Kaduna and Zaria was studied through nasal swabs collection by a one-stage cluster sampling and analysed using standard microbiological tests and genotyping methods. Questionnaires were also administered to assess for risk factors associated with MRSA carriage. Prevalence was 10% and 6.2% from the horses and horse handlers respectively. The S. aureus isolates showed highest resistance rates of 54.6% to penicillin and oxacillin. There were high resistance rates also to erythromycin, and tetracycline of 47.7% and 34.1% respectively. Resistance of 22.7% to amikacin and gentamicin, 6.8% to sulphamethoxazole+trimethoprim, 2.3% to ciprofloxacin, and 2.3% to chloramphenicol were also observed. Multidrug resistance (MDR) was found among 74.8% of the MRSA isolates. SCCmec typing showed types I, III, and IV in both the horses and the horse handlers while one horse was found to carry SCCmec V. Possible risk factors identified for MRSA carriage by horse handlers included being a veterinarian, exposure to antimicrobial agents, healthcare facility visitation, and personal hygiene. For the horses, risk factors from this study were allergy and wound management. MRSA nasal carriage in horses and horse handlers, as well as MDR (multidrug resistant) strains of S. aureus have been established from this study, this is of public health concern.

Biorisk Management and Antibiotic Susceptibility Pattern of Biofilm producing Pseudomonas aeruginosa Isolated from Broiler Chicken: A Public Health Concern

Control of biosecurity and biosecurity within poultry consists of a set of practical measures meant to prevent and control the spread of disease between people and animals. Infections, caused mainly by zoonotic agents, occur frequently due to the lack of safety monitoring regulations, as well as the inappropriate use of antimicrobial products, leading to the emergence of antimicrobial-resistant microorganisms. Pseudomonas aeruginosa, often known as the MDR pathogen has evolved resistance to multiple antibiotics. Because of its propensity to build biofilms in meat and other food products, P. aeruginosa is even more resilient to the phenomenon of drug resistance which is a major public health issue. Standard microbiological and biochemical tests were used to isolate and identify P. aeruginosa from a total of 100 meat samples (20 from each district from broiler chicken meat) gathered from various butcher shops and supermarkets. The Kirby Bauer method was used to identify antibiotic resistance, while the microtiter plate test was used to monitor biofilm formation. It was found that P. aeruginosa was identified from 22% of the broiler chicken meat samples and showed resistance to Cloxacillin, teicoplanin, ciprofloxacin, imipenem, and meropenem, followed by linezolid, streptomycin, amikacin, compound sulphonamide, aztreonam and cefepime which showed intermediate resistance. Multiple Antibiotic Resistance Index (MARI) was calculated as 0.45 for a total of 11 antibiotics. Also, all 22 MDR isolates of P. aeruginosa tested positive for the presence of the biofilm. In conclusion, it was determined that chicken meat was contaminated with Pseudomonas aeruginosa, and these strains that produce biofilms are more resistant to antibiotics. Thus, there is a serious threat to public health from biofilm-forming isolates found in broiler chickens.

Characterization of beta-lactamase producing Enterobacterales isolated from an urban community wastewater treatment plant in Iran.

he occurrence and characteristics of Extended Spectrum- and AmpC-β-lactamase producing Enterobacterales (ESBL-PE and AmpC-PE) in an urban wastewater treatment plant (WWTP) were investigated. A total of 30 wastewater samples were collected from all sections of WWTP. Enterobacterales were isolated and identified using standard microbiological tests. The antibiotic resistance profile was determined by the Kirby-Bauer disk diffusion method. Phenotypic screening for ESBL-PE and AmpC-PE isolates was performed by double-disk synergy and boronic acid disk potentiation tests, respectively. The isolates were examined for AmpC- and ESBL-encoding genes by PCR and sequencing methods. Among 146 Enterobacterales isolates, 8.9% (n=13) [ESBL-only; 5.48% (n=8) and ESBL + AmpC; 3.42% (n=5)] were ESBL-producers and 15.75% (n=23) [AmpC-only; 12.33% (n=18) and ESBL + AmpC; 3.42% (n=5)] AmpC-producers. Hafnia spp. with 33.33% (n=1/3) and E. coli with 20.58% (n=7/34) [ESBL-only; 17.64% (n=6/34) and ESBL + AmpC; 2.94% (n=1/34)] were the most common ESBL-producing bacteria. Enterobacter spp. with 37.50% (n=6/16) of isolates were the most common AmpC-producing organisms. ESBL- and/or AmpC-producing isolates were identified in all parts of the WWTP including 80% (n=8/10) of samples taken from effluent. Among ESBL-producing isolates, bla CTX-M , bla TEM, and bla SHV ESBL-encoding genes were found in 61.5% (n=8), 15.3% (n=2), and 7.7% (n=1) of isolates, respectively. All CTX-M-type enzymes belonged to the CTX-M-1 group and CTX-M-15 subgroup. bla TEM and bla SHV type genes belonged to bla TEM-20 and bla HSV-12 subtypes, respectively. bla DHA with 73.9% (n=17/23), and bla CIT and bla FOX with 30.4% (n=7/23) each, were the most common AmpC-encoding genes among AmpC-producing isolates. Overall, 75% of ESBL-producing and 55.5% of AmpC-producing isolates exhibited multi-drug resistance phenotypes. The organisms were most resistant against ampicillin (82.2%) nalidixic acid (43.8%) and cephalexin (41.1%). ESBL- and AmpC-producing Enterobacterales spp. with diverse genetic resistance backgrounds in WWTP effluent poses a significant risk to public health.

CRISPR Typing of Clinical Strains of Salmonella spp. Isolated in Tehran, Iran.

Salmonella is one of the most leading causes of food-born infection and death among infants and people with the poor immunity system. Because Salmonella spp. have diversity in the genome composition and pathogenicity, access to rapid identification and genotyping is necessary to control of salmonellosis. The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) typing is a genotyping method that checks these variable sequences in the bacterial genome in a specific species. This study aimed to differentiate Salmonella strains using CRISPR region. Salmonella isolates, previously identified via standard microbiological and molecular tests, were subjected to the study. Bacterial DNA was extracted and PCR was done using specific primers. The different PCR products were sequenced and the repeats patterns were used to identify additional or degenerate repeat clusters in the CRISPR region. All different sequences were analyzed using CRISPRtionary tool for dendrogram generation using the binary file. Overall, 119 strains of various Salmonella serovars were used. The result showed unique CRISPR and diversity in spacer both in sequence and the number. Analysis of the extracted sequence and band patterns illustrated that, except for S. infantis, both S. enteritidis and S. typhimurium isolates were classified as a separate cluster. CRISPR genotyping could provide serotype/spacers dictionary and it is performed at low cost and high speed in comparison to the other typing methods. Therefore, the assessment of CRISPR and spacer content can be considered as a powerful and practical discriminatory method for subtyping of Salmonella isolates.

Microbiological analysis of nosocomial pneumonia at Tanta University Chest Hospital

BackgroundHospital-acquired pneumonia (HAP) and ventilator-associated pneumonia (VAP) represent a major health problem among hospitalized patients leading to consequent morbidity and mortality specially after Covid-19 era and spread of multidrug-resistance organisms)MDRO) in hospitals.AimThis study aimed to analyze the commonest microorganisms responsible for HAP and VAP at Tanta University Chest Hospital.MethodsThis prospective observational study was done at Chest Department, Faculty of Medicine, started from June 2022 to February 2023. Fifty HAP patients’ sputum samples and 50 VAP patients (25 endotracheal aspirates and 25 bronchoalveolar lavages) were included. All collected samples were submitted to standard microbiological tests at Microbiology Department, Tanta Faculty of Medicine.ResultsA total number of 50 HAP and 50 VAP cases were included. Microbial isolates were relatively the same in both groups, where Klebsiella pneumoniae was the predominant isolates (56) followed by Staphylococcus aureus (25), Escherichia coli (14), Pseudomonas aeruginosa (13), Acinetobacter baumannii (5), Streptococcus pneumoniae (4), Enterococci (3), Stenotrophomonas maltophilia (2), Citrobacter freundii (2), Streptococcus pyogenes (2), Providencia stuartii (1), and 7 isolates of Candida. Antimicrobial susceptibility showed predominance of drug-resistance organisms in VAP (78%) versus HAP (28%), P-value: < 0.001. Klebsiella pneumonia showed higher rates of resistance (37 out of 56 isolates, P-value = 0.020). Four out of 5 Acinetobacter baumannii isolates were multidrug resistant, and 2 isolates of Stenotrophomonas maltophilia were polydrug resistant. Multivariate logistic regression analysis showed that intravenous antibiotic administration within last 3 months was associated with risk for MDR-HAP, while univariate regression analysis showed that intravenous antibiotic administration within the last 3 months was the strongest predictor for MDR-VAP. A higher mortality rate was recorded in VAP group (66%), versus (14%) in HAP group (P-value: < 0.001).ConclusionMicrobial isolates were relatively the same in both HAP and VAP, but VAP showed significant predominance of drug-resistant organisms. The most significant predictor for risk of MDRO infection in HAP and VAP was intravenous antibiotic administration within last 3 months; so, raising attention towards antibiotic choice may improve the outcome. Finally, VAP was associated with higher mortality compared to HAP.

Metagenomic sequencing for investigation of a national keratoconjunctivitis outbreak, Israel, 2022.

BackgroundEpidemics of keratoconjunctivitis may involve various aetiological agents. Microsporidia are an uncommon difficult-to-diagnose cause of such outbreaks.AimDuring the third quarter of 2022, a keratoconjunctivitis outbreak was reported across Israel, related to common water exposure to the Sea of Galilee. We report a comprehensive diagnostic approach that identified Vittaforma corneae as the aetiology, serving as proof of concept for using real-time metagenomics for outbreak investigation.MethodsCorneal scraping samples from a clinical case were subjected to standard microbiological testing. Samples were tested by calcofluor white staining and metagenomic short-read sequencing. We analysed the metagenome for taxonomical assignment and isolation of metagenome-assembled genome (MAG). Targets for a novel PCR were identified, and the assay was applied to clinical and environmental samples and confirmed by long-read metagenomic sequencing.ResultsFluorescent microscopy was suggestive of microsporidiosis. The most abundant species (96.5%) on metagenomics analysis was V. corneae. Annotation of the MAG confirmed the species assignment. A unique PCR target in the microsporidian rRNA gene was identified and validated against the clinical sample. The assay and metagenomic sequencing confirmed V. corneae in an environmental sludge sample collected at the exposure site.ConclusionsThe real-time utilisation of metagenomics allowed species detection and development of diagnostic tools, which aided in outbreak source tracking and can be applied for future cases. Metagenomics allows a fully culture-independent investigation and is an important modality for public health microbiology.

Detection of Extended Spectrum Beta Lactamase Producing Escherichia Coli and Klebsiella Species Isolated from Urine Samples of UTI Patients by Phenotypic and Genotypic Methods

Background: Escherichia coli and Klebsiella spp. are the most common organisms causing urinary tract infection (UTI) and commonly responsible for extended spectrum beta lactamase (ESBL) production. This study was carried out to detect ESBL producing uropathogenic Escherichia coli and Klebsiella spp. by phenotypic and genotypic method. Rapid and accurate detection of ESBL producing E. coli and Klebsiella spp. has an important role to avoid treatment failure. Materials and Methods: This was a cross sectional observational study and carried out in department of microbiology in Chittagong Medical College, Bangladesh from January to December 2017. Urine was collected from suspected UTI patients and standard microbiological and biochemical tests were carried out. ESBL producing E. coli and Klebsiella spp. were identified by phenotypic confirmatory disc diffusion test (PCDDT). Polymerase chain reaction (PCR) was performed by using standard protocol with specific primers. Results: 448 urine samples were collected. Among them, 140 showed bacterial growth; 72 were E. coli and 35 were Klebsiella spp. Among E. coli 38(52.8%) and in Klebsiella spp. 15(42.9%) were detected as ESBL producers by PCDDT respectively. Among E. coli, 41(56.9%) and in Klebsiella spp. 21(60%) strains produced ESBL genes by PCR. Out of 38 phenotypically positive E. coli, 7 strains do not carry any detectable genes. Similarly, out of 15 phenotypically positive Klebliella spp. 3 isolates did not produce any detectable gene. On the other hand, 10 E. coli isolates and 9 Klebsiella spp. carry detectable genes although these were not phenotypically ESBL producers. Moreover, ESBL producing E. coli and Klebsiella spp. showed more multidrug resistant than Non-ESBL producing E. coli and Klebsiella spp. Conclusion: This study revealed that large portion of E. coli and Klebsiella spp. was ESBL producers. PCR can detect some additional cases of ESBL producing isolates. So, PCR can be used along with phenotypic ......

Are we missing the silver lining of COVID-19 pandemic: An analytical study to determine effects of three COVID-19 peaks on antimicrobial susceptibility of Staphylococcus aureus isolates

ABSTRACT Background: Given the evolving nature of COVID-19, for better understanding of its effect on antimicrobial resistance of Staphylococcus aureus (S. aureus), it becomes crucial that we follow the resistance patterns across different surges of COVID-19 cases. Methods: This prospective surveillance study extended over two years from January 2020-March 2022 and was conducted in a healthcare center of North India. Susceptibility patterns of Staphylococcus aureus during January-March 2020 were considered as prepandemic patterns. Processing of clinical specimens, identification of S. aureus, and in-vitro antibiotic susceptibility testing were performed in accordance with standard microbiological testing procedures and Clinical Laboratory Standard Institute guidelines. Results: Lowest prevalence (38.9%) of Methicillinresistant S. aureus was reported during January-March 2021 and July-September 2021. More than 50% S. aureus isolates were susceptible to linezolid, cotrimoxazole, tetracycline, and gentamicin in January-March 2020. In January-March 2021, ≥50% of S. aureus isolates from clinical specimens were additionally susceptible to clindamycin and erythromycin. Antibiotic agents of linezolid, tetracycline, clindamycin, and cotrimoxazole were susceptible in ≥50% of S. aureus isolates in January-March 2022. Conclusions: This study reveals a sharp decline in overall resistance to commonly prescribed antibiotic agents for S. aureus isolates after first peak of COVID-19 cases. However, same trend was not observed in subsequent peaks and probably we are approaching the same resistance levels that were seen prior to COVID-19 pandemic.

Clinical metagenomic sequencing of plasma microbial cell-free DNA for febrile neutropenia in patients with acute leukaemia

ObjectivesTo evaluate the diagnostic performance and clinical impact of metagenomic next-generation sequencing (mNGS) of plasma microbial cell-free DNA (mcfDNA) in febrile neutropenia (FN). MethodsIn a 1-year, multicentre, prospective study, we enrolled 442 adult patients with acute leukaemia with FN and investigated the usefulness of mNGS of plasma mcfDNA for identification of infectious pathogens. The results of mNGS were available to clinicians in real time. The performance of mNGS testing was evaluated in comparison with blood culture (BC) and a composite standard that incorporated standard microbiological testing and clinical adjudication. ResultsIn comparison with BC, the positive and negative agreements of mNGS were 81.91% (77 of 94) and 60.92% (212 of 348), respectively. By clinical adjudication, mNGS results were categorized by infectious diseases specialists as definite (n = 76), probable (n = 116), possible (n = 26), unlikely (n = 7), and false negative (n = 5). In 225 mNGS-positive cases, 81 patients (36%) underwent antimicrobials adjustment, resulting in positive impact on 79 patients and negative impact on two patients (antibiotics overuse). Further analysis indicated that mNGS was less affected by prior antibiotics exposure than BC. DiscussionOur results indicate that mNGS of plasma mcfDNA increased the detection of clinically significant pathogens and enabled early optimization of antimicrobial therapy in patients with acute leukaemia with FN.

Profile of Cutaneous Bacterial Flora in Pemphigus Patients.

Objectives  Pemphigus, a group of autoimmune bullous diseases, can be fatal, resulting from overwhelming opportunistic infection of lesions secondary to cutaneous bacterial infections. This study aimed to look into the cutaneous bacterial infection profile of pemphigus patients as timely identification and appropriate treatment can play a major role in reducing mortality. Materials and Methods  Pus samples/swabs received from patients with pemphigus over a 2-year period from July 2018 to June 2020 were subjected to standard microbiological culture techniques and susceptibility testing. The frequency of isolation and susceptibility profile of the different bacterial pathogens toward various antimicrobial agents were interpreted and analyzed as per the Clinical and Laboratory Standards Institute's guidelines. Results  Samples from 315 patients were received during the study period comprising of 203 (64.4%) males and 112 (35.5%) females. Of 211 samples which were culture-positive, a total of 245 bacterial isolates were obtained, comprising of 158 Gram-positive cocci and 87 Gram-negative bacilli. Staphylococcus aureus (138, 56.3%) was the most common isolate followed by Pseudomonas aeruginosa (41, 16.7%) and Escherichia coli (16, 6.5%). Methicillin resistance was observed in 24.6% Staphylococcus aureus isolates and carbapenem resistance in 9.5 to 14.6% Gram-negative bacilli. Conclusions  Study findings emphasize the need for continuous monitoring of cutaneous pemphigus lesions for appropriate choice of antimicrobial therapy.

Detection of Mycoplasma gallisepticum, Isolation, and Determination of Tylosin Susceptibility of Isolates from Commercial Chickens.

Mycoplasma gallisepticum (MG) is a contagious avian pathogen that causes financial losses to the poultry industry. Isolation of the pathogen is difficult and time-consuming, and therefore, far from a routine method. Serological testing methods to detect antibodies resistant to MG are widely used in routine diagnosis. Tylosin is a class of macrolide antibiotics tremendously administered in veterinary medicine for the treatment of mycoplasmosis and prophylaxis. This study aimed to detect MG by immunoassay testing, culture, and polymerase chain reaction (PCR) in commercial poultry farms and to investigate the tylosin susceptibility of the isolates. To verify the presence of antibodies resistant to MG, 750 blood samples were randomly collected from 38 broiler farms from 2019 to 2022 in Mazandaran and Golestan provinces, Iran, and rapid slide agglutination (RSA) assay was performed. Positive results were analyzed by the enzyme-linked immunosorbent assay (ELISA) for further investigation. Here, 920 swab samples were collected from 38 non-vaccinated commercial farms for culture, and PCR tests were performed for the isolated strains. The activities of tylosin were tested in vitro against these isolates using the broth microdilution method. The lowest antibiotic concentration that resulted in a color change was considered the minimum inhibitory concentration (MIC) value. Twenty-four (63.1%) farms were positive in the RSA test, and 21 (55.2%) farms were positive in the ELISA test. Nine (23.68%) of the farms grew on culture media, and 8 (21.05%) were detected as Gallisepticum species by PCR. The geometric mean of MIC for tylosin was 5.75 µg/ml, MIC50 was 4 µg/ml, and MIC90 was 8 µg/ml. The results indicated that commercial farms were infected with MG. Considering the ability of MG to spread and the probable use of the RSA test as a rapid and cheap method, it can be argued that ELISA and RSA serological tests can be used to find MG in poultry flocks, and the positive result should be confirmed by standard microbiological tests or PCR. It was also found that the isolated parts of MG changed their sensitivity to tylosin, indicating the need for routine testing to optimize treatment dose and efficiency.

Molecular detection of plasmid-mediated quinolone resistance genes among clinical isolates of Klebsiella pneumoniae during Covid-19 pandemic

Plasmid-mediated quinolone resistance (PMQR) genes confer low resistance to Fluoroquinolones (FQs). This study aims to detect five PMQR genes among FQs-resistant Klebsiella pneumoniae isolated from various clinical specimens. Out of 120 K. pneumoniae isolates, 68 FQs-resistance K. pneumoniae were included in a molecular study. Standard microbiological tests were used for identification and antimicrobial susceptibility. For the detection of PMQR genes, conventional polymerase chain reaction was used. A molecular study revealed that (73.5%) of samples harbored PMQR genes, and among them, 58% were co-carriages of PMQR gene variants. Aac (6’)-Ib-cr gene was predominant (47.1%) among samples, and qepA had the lowest percentage (11.8%), qnr genes were (32.4%) (29.4%) (20.6%) qnrS, qnrB, and qnrA respectively. Overall, high percentages of PMQR genes were detected, and almost all of samples were phenotypically resistant to ciprofloxacin. As well, there was a significant statistical relationship between phenotypically ESBL-producers and qnrB and qepA genes.

A pilot study to develop assessment tools for Group A Streptococcus surveillance studies.

Group A Streptococcus (GAS) causes pharyngitis (sore throat) and impetigo (skin sores) GAS pharyngitis triggers rheumatic fever (RF) with epidemiological evidence supporting that GAS impetigo may also trigger RF in Australian Aboriginal children. Understanding the concurrent burden of these superficial GAS infections is critical to RF prevention. This pilot study aimed to trial tools for concurrent surveillance of sore throats and skins sore for contemporary studies of RF pathogenesis including development of a sore throat checklist for Aboriginal families and pharynx photography. Yarning circle conversations and semi-structured interviews were performed with Aboriginal caregivers and used to develop the language and composition of a sore throat checklist. The sore throat story checklist was combined with established methods of GAS pharyngitis and impetigo surveillance (examination, bacteriological culture, rapid antigen detection and serological tests) and new technologies (photography) and used for a pilot cross-sectional surveillance study of Aboriginal children attending their health clinic for a routine appointment. Feasibility, acceptability, and study costs were compiled. Ten Aboriginal caregivers participated in the sore-throat yarning circles; a checklist was derived from predominant symptoms and their common descriptors. Over two days, 21 Aboriginal children were approached for the pilot surveillance study, of whom 17 were recruited; median age was 9 years [IQR 5.5-13.5], 65% were female. One child declined throat swabbing and three declined finger pricks; all other surveillance elements were completed by each child indicating high acceptability of surveillance assessments. Mean time for screening assessment was 19 minutes per child. Transport of clinical specimens enabled gold standard microbiological and serological testing for GAS. Retrospective examination of sore throat photography concorded with assessments performed on the day. Yarning circle conversations were effective in deriving culturally appropriate sore throat questionnaires for GAS pharyngitis surveillance. New and established tools were feasible, practical and acceptable to participants and enable surveillance to determine the burden of superficial GAS infections in communities at high risk of RF. Surveillance of GAS pharyngitis and impetgio in remote Australia informs primary RF prevention with potential global translation.