Routine Microbiological Examination Research Articles

The diagnostic value of metagenomics next-generation sequencing in HIV-infected patients with suspected pulmonary infections.

Traditional microbiological detection methods used to detect pulmonary infections in people living with HIV (PLHIV) are usually time-consuming and have low sensitivity, leading to delayed treatment. We aimed to evaluate the diagnostic value of metagenomics next-generation sequencing (mNGS) for microbial diagnosis of suspected pulmonary infections in PLHIV. We retrospectively analyzed PLHIV who were hospitalized due to suspected pulmonary infections at the sixth people hospital of Zhengzhou from November 1, 2021 to June 30, 2022. Bronchoalveolar lavage fluid (BALF) samples of PLHIV were collected and subjected to routine microbiological examination and mNGS detection. The diagnostic performance of the two methods was compared to evaluate the diagnostic value of mNGS for unknown pathogens. This study included a total of 36 PLHIV with suspected pulmonary infections, of which 31 were male. The reporting period of mNGS is significantly shorter than that of CMTs. The mNGS positive rate of BALF samples in PLHIV was 83.33%, which was significantly higher than that of smear and culture (44.4%, P<0.001). In addition, 11 patients showed consistent results between the two methods. Futhermore, mNGS showed excellent performance in identifying multi-infections in PLHIV, and 27 pathogens were detected in the BALF of 30 PLHIV by mNGS, among which 15 PLHIV were found to have multiple microbial infections (at least 3 pathogens). Pneumocystis jirovecii, human herpesvirus type 5, and human herpesvirus type 4 were the most common pathogen types. For PLHIV with suspected pulmonary infections, mNGS is capable of rapidly and accurately identifying the pathogen causing the pulmonary infection, which contributes to implement timely and accurate anti-infective treatment.

Distribution of MIC values of antibacterial drugs for Flavobacteriales isolated from respiratory samples in Russian patients with cystic fibrosis

Objective. Analysis of the distribution of the values of the minimum inhibitory concentrations (MIC) of a number of antibacterial drugs in relation to representatives of the order Flavobacteriales isolated from respiratory samples from patients with cystic fibrosis of the Russian Federation by the method of double serial dilutions. Materials and Methods. The distribution of the values of MIC of a number of antibacterial drugs was evaluated in relation to 100 strains of bacteria, representatives of the order Flavobacteriales, isolated from respiratory samples from patients with cystic fibrosis from 60 regions of the Russian Federation as part of a routine microbiological examination. Of these, 75 representatives of Chryseobacterium spp., among which C. arthrospherae – 28, C. formosense – 1, C. gambrini – 3, C. gleum – 10, C. indologenes – 20, C. joostei – 1, C. oraniemense – 10, C. shandongense – 2 strains, 4 strains of Elizabethkingia spp., among which 2 – E. miricola, and one strain of E. meningoseptica and E. anopheles, respectively, as well as 21 strains of E. falsenii. Identification of all isolated cultures was carried out using MALDI-ToF mass spectrometry (Bruker, Germany). The MIC values were determined for 17 antimicrobial drugs: amikacin, amoxicillin/clavulanate, aztreonam, cefotaxime, ceftazidime, ceftazidime/avibactam, ceftolozane/ tazobactam, ciprofloxacin, colistin, ertapenem, gentamicin, imipenem, meropenem, piperacillin/ tazobactam, tigecycline, tobramycin and trimethoprim/sulfamethoxazole using Sensititre kits DKMGN. Results. The MIC values of colistin, cefotaxime and tobramycin more than 8 µg/ml, as well as more than 2 µg/ml with respect to ertapenem was demonstrated for 100% of isolates. For most strains, the MIC values of imipenem and meropenem were more than 4 µg/ml. MIC of ceftazidime against 20% of Chryseobacterium spp. strains was up to 2 µg/ml. Indicators of MIC of amikacin in relation to Elizabethkingia spp. strains were 32 µg/ml or more. For 38% of the strains of Chryseobacterium spp. and E.falsenii, this value did not exceed 8 µg/ml. As in the case of amikacin, all Elizabethkingia spp. strains demonstrated high levels of gentamicin MIC – 8 µg/ml (25% of strains) and more (75% of strains). For 20% of all tested strains, the MIC value was in the range of ≤ 2 µg/ml. In relation to half of the tested isolates, the MIC values for ceftazidime/avibactam, as well as ceftolozane/tazobactam were at the level of up to 2⁄4 µg/ml inclusive. More than a third of the strains had a level of MIC ciprofloxacin up to 0.25 µg/ml inclusive. For 25% of strains, the level of MIC of tigecycline was up to 0.5 µg/ml, inclusive, the lowest MIC indicators for the tested group of strains were demonstrated for trimethoprim/sulfamethoxazole: for 88% of strains, the MIC value was ≤ 1⁄19 µg/ml, for another 9% of strains, this indicator was 2⁄38 µg/ml. Conclusions. Representatives of the order Flavobacteriales are a group of microorganisms characterized by multiple antibiotic resistance. Most strains isolated from patients with cystic fibrosis in the Russian Federation retain low MIC values for trimethoprim/sulfamethoxazole.

Clinical performance of metagenomic next-generation sequencing for the rapid diagnosis of talaromycosis in HIV-infected patients.

Talaromycosis is an invasive endemic mycosis caused by the dimorphic fungus Talaromyces marneffei (T. marneffei, TM). It mainly affects immunodeficient patients, especially HIV-infected individuals, which causes significant morbidity and mortality. Culture-based diagnosis takes a long turnaround time with low sensitivity, leading to treatment delay. In this study, we aimed to evaluate the performance of Metagenomic Next-Generation Sequencing (mNGS) for the rapid diagnosis of talaromycosis in HIV-infected patients. Retrospectively analysis was conducted in HIV-infected cases at Changsha First Hospital (China) from January 2021 to March 2022. Patients who underwent routine microbiological examination and mNGS testing in parallel were enrolled. The clinical final diagnosis was used as a reference standard, and cases were classified into the TM group (60 cases) and the non-TM group (148 cases). The clinical performances of mNGS were compared with culture and serum Galactomannan (GM). The mixed infections detected by mNGS were analyzed. The impact of mNGS detection on treatment was also investigated. The sensitivity of mNGS test reached 98.3% (95% CI, 89.8-99.9), which was significantly higher than culture (66.7% [95% CI, 53.2-77.9], P < 0.001) and serum GM (83.3% [95% CI, 71.0-91.2], P < 0.05). The specificity of 98.6% (95% CI, 94.7-99.7) was similar to culture (100.0% [95% CI, 96.8-100.0], P = 0.156), and superior to serum GM (91.9% [95% CI, 85.9-95.5], P < 0.05). In bronchoalveolar lavage fluid (BALF) samples, the positive rate of mNGS was 97.6%, which was significantly higher than culture (28.6%, P <0.001). mNGS has excellent performance in the identification of mixed infection in TM group patients. Cytomegalovirus, Epstein-Barr virus and Pneumocystis jirovecii were the most common concurrent pathogens. In summary, 60.0% (36/60) patients were added or adjusted to antimicrobial therapy after mNGS test. mNGS is a powerful technique with high specificity and sensitivity for the rapid diagnosis of talaromycosis. mNGS of BALF samples may be a good option for early identification of T. marneffei in HIV-infected individuals with manifestations of infection. Moreover, mNGS shows excellent performance in mixed infection, which benefits timely treatment and potential mortality reduction.

Application of metagenomic next-generation sequencing in the detection of pathogens in bronchoalveolar lavage fluid of infants with severe pneumonia after congenital heart surgery.

BackgroundMetagenomic next-generation sequencing (mNGS) has become a valuable diagnostic tool in clinical etiology detection due to its rapidity, accuracy, and high throughput. However, the role of this technology in the diagnosis and treatment of infants with severe pneumonia after congenital heart surgery is still unclear.MethodsWe conducted a retrospective cohort study of infants with severe pneumonia after congenital heart surgery. Samples were collected from infants in the hospital’s cardiac intensive care unit between January 2010 and January 2022. The conventional microbiological test (CMT) group consisted of patients who underwent routine microbiological examination, and the infants’ bronchoalveolar lavage fluid was examined. The mNGS group consisted of patients who underwent mNGS and routine microbiological examinations.ResultsThe overall positive rate of mNGS was significantly higher than that of CMT (88.4 vs. 62.5%, P = 0.009). After receipt of the microbiological results, 30/43 (70%) patients in the mNGS group had a change in antibiotic use compared with 14/40 (35%) in the CMT group (P = 0.002). Subsequently, after adjusting the treatment plan according to the microbiological test results, the number of people with improved pulmonary infection in the mNGS group was significantly higher than that in the CMT group (63 vs. 28%, P < 0.05). In addition, the duration of invasive ventilation, length of CICU stay and total hospital length of stay in the mNGS group were significantly lower than those in the CMT group (P < 0.05).ConclusionmNGS is a valuable tool to determine the etiology of infants with severe pneumonia after congenital heart disease surgery. It can significantly improve the sensitivity of pathogen detection, which can help determine appropriate antimicrobial drugs, improve the diagnostic accuracy of the disease, and improve outcomes.

Kültürü Yapılan Deniz Balıklarında Karaciğer Yağlanması ve Bakteriyel Koenfeksiyon

In the present study, we sampled sea bream and sea bass, weighing 400-450g, for routine microbiological, hematological, macroscopical and histopathological examinations. Samples were taken from seemingly healthy fish, which displayed increased feed conversion rates, signs of cold shock and slow movement. Routine bacteriological analyses included basic microbiological analyses for oxidase and catalase activity, Gram staining and O/F fermentation using API test kits. Furthermore, 16S rRNA gene sequencing was performed for identification. Serum biochemical and hematological values were determined using a Vetscan®VS2 analyzer and blood smears. Internal organs were examined by routine histopathological techniques. Hepatic fat accumulation and necrosis were noted, and liver damage was observed to be associated with significant alterations in liver enzymes. Due to multi-organ dysfunction and serious hematological disorders, primarily Vibrio alginolyticus also Alteromonas and Pseudoalteromonas species were isolated from the affected fish and co-infections were detected.

Fatty Liver Disease and Bacterial Co-Infection in Cultured Marine Fish

In the present study, we sampled sea bream and sea bass, weighing 400-450g, for routine microbiological, hematological, macroscopical and histopathological examinations. Samples were taken from seemingly healthy fish, which displayed increased feed conversion rates, signs of cold shock and slow movement. Routine bacteriological analyses included basic microbiological analyses for oxidase and catalase activity, Gram staining and O/F fermentation using API test kits. Furthermore, 16S rRNA gene sequencing was performed for identification. Serum biochemical and hematological values were determined using a Vetscan®VS2 analyzer and blood smears. Internal organs were exam-ined by routine histopathological techniques. Hepatic fat accumulation and necrosis were noted, and liver damage was observed to be associated with significant alterations in liver enzymes. Due to multi-organ dysfunction and serious hematological disorders, primarily Vibrio alginolyticus also Alteromonas and Pseudoalteromonas species were isolated from the affected fish and co-infections were detected.

Comparative genome analysis of colistin-resistant OXA-48-producing Klebsiellapneumoniae clinical strains isolated from two Iranian hospitals

BackgroundCarbapenemase-producing Klebsiella pneumoniae (CP-KP) is becoming extensively disseminated in Iranian medical centers. Colistin is among the few agents that retains its activity against CP-KP. However, the administration of colistin for treatment of carbapenem-resistant infections has increased resistance against this antibiotic. Therefore, the identification of genetic background of co-carbapenem, colistin-resistance K.pneumoniae (Co-CCRKp) is urgent for implementation of serious infection control strategies.MethodsFourteen Co-CCRKp strains obtained from routine microbiological examinations were subjected to molecular analysis of antimicrobial resistance (AMR) using whole genome sequencing (WGS).ResultsNine of 14 K.pneumoniae strains belonged to sequence type (ST)-11 and 50% of the isolates had K-locus type 15. All strains carried blaOXA-48 except for P26. blaNDM-1 was detected in only two plasmids associated with P6 and P26 strains belonging to incompatibility (Inc) groups; IncFIB, IncHI1B and IncFII. No blaKPC, blaVIM and blaIMP were identified. Multi-drug resistant (MDR) conjugative plasmids were identified in strains P6, P31, P35, P38 and P40. MICcolistin of K. pneumoniae strains ranged from 4 to 32 µg/ml. Modification of PmrA, PmrB, PhoQ, RamA and CrrB regulators as well as MgrB was identified as the mechanism of colistin resistance in our isolates. Single amino acid polymorphysims (SAPs) in PhoQ (D150G) and PmrB (R256G) were identified in all strains except for P35 and P38. CrrB was absent in P37 and modified in P7 (A200E). Insertion of ISKpn72 (P32), establishment of stop codon (Q30*) (P35 and P38), nucleotides deletion (P37), and amino acid substitution at position 28 were identified in MgrB (P33 and P42). None of the isolates were positive for plasmid-mediated colistin resistance (mcr) genes. P35 and P38 strains carried iutA, iucD, iucC, iucB and iucA genes and are considered as MDR-hypervirulent strains. P6, P7 and P43 had ICEKp4 variant and ICEKp3 was identified in 78% of the strains with specific carriage in ST11.ConclusionIn our study, different genetic modifications in chromosomal coding regions of some regulator genes resulted in phenotypic resistance to colistin. However, the extra-chromosomal colistin resistance through mcr genes was not detected. Continuous genomic investigations need to be conducted to accurately depict the status of colistin resistance in clinical settings.

Microbial profile of corneal ulcer in a tertiary eye care hospital at Tamil Nadu

Purpose: To identify the most common etiological agent in corneal ulceration in a tertiary care eye hospital.Materials and Methods: A total of 50 patients with suspected infectious corneal ulcers presenting to ophthalmology out-patient department in Rajah Muthiah Medical College and Hospital were evaluated. Sociodemographic data and information pertaining to risk factors were recorded. All patients were examined and corneal cultures and scrapings were performed.Results: Of 50 patients microbiological etiology was established in 39 cases (78%). Of these 36(72%) were male. Of 39 positive cases 42% were fungi and 36% were bacterial and 22% showed no growth. The most common isolated fungus was fusarium (67%) followed by aspergillus (33%). Streptococci was the most common isolated bacteria.Conclusion: Routine microbiological examination of patients with corneal ulcer is necessary to analyze the changing trends of the etiology.

Bacterial enteropathogens causing acute diarrhoea in children in a tertiary care hospital, Imphal

Background: Diarrhoeal diseases are responsible for causing 3 million deaths worldwide every year especially among the children and also the commonest cause of morbidity and mortality in developing countries like India. Infective diarrhoea could be either bacterial, viral, parasitic or occasionally a combination of these.Methods: A cross-sectional study was carried out in children below 12 years with acute diarrhoea in theMicrobiology Department, RIMS, Imphal for a period of 2 years. Stool samples were subjected to routine microbiological examination, followed by culture and sensitivity. Data were collected in a predesigned data collection sheet.Results: Majority of the diarrhoeal cases were seen among the age group of 1-3 years (44.3%), predominantly among the male children (66.2%) and mostly in summer. Out of 210 culture positive stool samples, Escherichia coli(83.3%) was the predominant enteropathogen with followed by Shigella spp.(12.9%), Klebsiella spp. (2.9%) and Salmonella spp. (1%). Serotyping revealed thirty five enteropathogenic E. coli, eighteen Shigella flexneri, seven Shigellasonnei, two Shigella boydii and two Salmonella typhimurium. Majority of the isolates showed high resistance to amoxicillin, ampicillin, ciprofloxacin, ofloxacin and cotrimoxazole.Conclusions: Bacterial enteropathogens are an important cause of acute diarrhoea among children. Rehydration therapy remains the initial treatment. Though it is usually self-limiting, empirical and specific antimicrobial therapy can be considered in certain situations. Awareness of improving hygiene and infectious diseases may help reduce the burden of infection.

Detection of Various Streptococcus spp. and Their Antimicrobial Resistance Patterns in Clinical Specimens from Austrian Swine Stocks.

Knowledge of pathogenic potential, frequency and antimicrobial resistance patterns of porcine Streptococcus (S.) spp. other than S. suis is scarce. Between 2016 and 2020, altogether 553 S. spp. isolates were recovered from clinical specimens taken from Austrian swine stocks and submitted for routine microbiological examination. Antimicrobial susceptibility testing towards eight antimicrobial substances was performed using disk diffusion test. All isolates from skin lesions belonged to the species S. dysgalactiae subspecies equisimilis (SDSE). S. hyovaginalis was mainly isolated from the upper respiratory tract (15/19) and S. thoraltensis from the genitourinary tract (11/15). The majority of S. suis isolates were resistant to tetracycline (66%), clindamycin (62%) and erythromycin (58%). S. suis isolates from the joints had the highest resistance rates. S. suis and SDSE isolates resistant to tetracycline were more likely to be resistant to erythromycin and clindamycin (p < 0.01). Results show that different species of Streptococcus tend to occur in specific body sites. Nevertheless, a statement whether these species are colonizers or potential pathogens cannot be given so far. High resistance rates of S. suis towards tetracyclines and erythromycin and high recovery rates of S. suis from lung tissue should be considered when treating pigs with respiratory diseases.

Lean back and wait for the alarm? Testing an automated alarm system for nosocomial outbreaks to provide support for infection control professionals.

IntroductionOutbreaks of communicable diseases in hospitals need to be quickly detected in order to enable immediate control. The increasing digitalization of hospital data processing offers potential solutions for automated outbreak detection systems (AODS). Our goal was to assess a newly developed AODS.MethodsOur AODS was based on the diagnostic results of routine clinical microbiological examinations. The system prospectively counted detections per bacterial pathogen over time for the years 2016 and 2017. The baseline data covers data from 2013–2015. The comparative analysis was based on six different mathematical algorithms (normal/Poisson and score prediction intervals, the early aberration reporting system, negative binomial CUSUMs, and the Farrington algorithm). The clusters automatically detected were then compared with the results of our manual outbreak detection system.ResultsDuring the analysis period, 14 different hospital outbreaks were detected as a result of conventional manual outbreak detection. Based on the pathogens’ overall incidence, outbreaks were divided into two categories: outbreaks with rarely detected pathogens (sporadic) and outbreaks with often detected pathogens (endemic). For outbreaks with sporadic pathogens, the detection rate of our AODS ranged from 83% to 100%. Every algorithm detected 6 of 7 outbreaks with a sporadic pathogen. The AODS identified outbreaks with an endemic pathogen were at a detection rate of 33% to 100%. For endemic pathogens, the results varied based on the epidemiological characteristics of each outbreak and pathogen.ConclusionAODS for hospitals based on routine microbiological data is feasible and can provide relevant benefits for infection control teams. It offers in-time automated notification of suspected pathogen clusters especially for sporadically occurring pathogens. However, outbreaks of endemically detected pathogens need further individual pathogen-specific and setting-specific adjustments.

Auditing the Routine Microbiological Examination of Pus Swabs From Uncomplicated Perianal Abscesses: Clinical Necessity or Old Habit?

BackgroundObtaining pus swabs from perianal abscesses after incision and drainage for subsequent microbiological analysis is traditionally performed by general surgeons. Our aim is to assess the current practice in our institution, emphasizing on whether pus swabs were sent or not, as well as to identify any associations between the revealed microbiology and the occurrence of immediate post-operative complications and re-admission rates with fistula-in-ano up to 12 months post the emergency drainage. Finally, we aimed to identify if the any members of the surgical team reviewed at any stage post-operatively the results of the microbiological examination of the obtained pus swabs and if that resulted in changes of the patient management.MethodsWe reviewed the operative findings and perioperative antimicrobial management of all patients within our institution that required surgical treatment of perianal abscesses over a 6-week period and re-assessed them after 12 months from the performed drainage, with respect to re-admission and identification of occurred fistula-in-ano.ResultsA total of 24 patients met our inclusion criteria. Pus swabs were sent in 66.7% of cases and only a third of the requested microbiology reports were reviewed by a part of the surgical team. All patients were discharged prior to the release of the microbiology results with no subsequent change in the management plan. We did not find any consistent association between the microbiology results and re-admission with perianal abscess, with or without fistula-in-ano.ConclusionsWe do not recommend routine use of pus swabs when draining perianal abscesses unless clinical concerns arise, including recurrent perianal sepsis, immuno-compromised status or extensive soft tissue necrosis, especially when these features are associated with systemic sepsis.

Determination of Beta-Lactamase Inactivation of Cephalexin by Validated RP-HPLC Method

Determination of the needed amount of liquid sterile Lactamator™ to be mixed to cephalexin, β-lactam antibiotic, for optimum deactivation of its molecule's antibacterial properties was conducted using RP-HPLC method. RP-HPLC method was validated for the parameters as linearity, accuracy, LOD, LOQ, and precision. Before the routine microbiological examination for any pharmaceutical dosage form containing β-lactam antibiotic, it is a must to make inactivate of β-lactam active pharmaceutical ingredient (API) by mixing with beta-lactamase before testing. So, the study indicated that mixing of 0.5 ml liquid sterile Lactamator™ with phosphate buffer solution pH (7.2) containing 50 mg cephalexin, with holding the test sample for 90 minutes prior to HPLC measurement will deactivation of cefalexin molecule's antibacterial properties.

Quality of coroner's post-mortems in a UK hospital

The aim of this paper was, principally, to look at the coroner's post-mortem report quality regarding adult medical patients admitted to an English hospital; and to compare results with Royal College of Pathologists guidelines. Hospital clinical notes of adult medical patients dying in 2011 and who were referred to the coroner's office to determine the cause of death were scrutinised. Their clinical care was also reviewed. There needs to be a comprehensive approach to coroner's post-mortems such as routinely taking histological and microbiological specimens. Acute adult medical patient care needs to improve. Steps should be taken to ensure that comprehensive coroner's post-mortems are performed throughout the UK, including with routine histological and microbiological specimens examination. Additionally, closer collaboration between clinicians and pathologists needs to occur to improve emergency adult medical patient clinical care. The study highlights inadequacies in coroner's pathology services.

DETECTION OF &lt;i&gt;MYCOPLASMA HOMINIS&lt;/i&gt; IN PATIENTS WITH URINARY TRACT INFECTIONS

Mycoplasma hominis is one of the well known genital mycoplasma agent that has been recognized as potentially pathogenic species and associated with various infectious diseases in adult men and woman. The organism could cause pelvic inflammatory disease, postpartum, postabortion fever and pyelonephritis. The Aim of this prospective study was to detemine the occurrence and prevalence of Mycoplasma hominis in patients with urinary tract infections, since they are usually not detected by routine microbiological examination of urine samples in laboratory department. For this purpose, Culture method was to be attempted as the current laboratory methods of choice for detection urogenital mycoplasmas infections. This study was designed as a case-control study in which a total of 100 patients with mean age of the of 42.78±22.49 (95% CI = 39-49.17) in case group as well as a mean age of 48.18±18.81 ((95% CI = 42.83-53.53) in the control group. The patients of both sexes were investigated for the presence of M. hominis and they were admitted to the urology department at asser central hospital in Abha (a city in southwest region of Saudi Arabia), during over a period of one year (February, 2013-March, 2014). A detailed history with specific urinary symptoms and signs was obtained as a result. These patients were divided into two groups: The first group (or case group) and the second group (or control group): The case group, included 50 symptomatic patients who had been admitted in hospital and they were examined for the presence of one or more of the following specific urinary symptoms and signs: Urinary frequency, dysuria, suprapubic/pelvic pain. Unlike the case group, the control group included 50 asymptomatic patients who were free of any specific urinary symptoms and they were matched by the first group for their age index. First voided urine specimens were tested using urine microscopy, culture for M hominis was performed and the isolates were identified serologically by growth inhibition test (disc method). In the case group, 8 cases out of 50 and in control group 1 out 50 were positive for M. hominis infection. The incidence of M. hominis was higher and statistically significant among case symptomatic patients group (8/50, 16%) than control asymptomatic patients group patients (1/50, 2%). On applying tests of significance to the observed data for isolation of Mycolplasma hominis among cases and controls, the values were found to statistically significant. Chi-square test with Yate’s correction [χ2 4.396; p value 0.036] and the Fisher’s exact test (2 tailed) [p value = 0.0309] indicated the relevance of the findings. The Relative Risk (RR) for the occurrence of Mycoplasma hominis infection was estimated to be 8 (95% C.I = 1.0386 -61.6235). The odds ratio was determined to be 0.1071 (95% CI = 0.129-0.892) and the risk ratio was 0.2063 (95% CI = 0.0322-1.3224). This is the first report study for the detection of M hominis in patients with urinary tract infections performed in Saudi Arabia. Infection caused by M hominis was associated with higher incidence rate in patients with symptomatic urinary tract diseases. Further work studies is needed for their rapid detection of these organism in urine samples using other diagnostic methods such as PCR. Further knowledge of the role and the effect of this pathogen in patients with urinary tract infections can be a prospective field of study.

Identification and characterization of methicillin-resistant Staphylococcus aureus (MRSA) from Austrian companion animals and horses

The aim of this study was to investigate the antimicrobial resistance, resistance gene patterns and genetic relatedness of a collection of Austrian methicillin-resistant Staphylococcus aureus (MRSA) isolates from companion animals and horses.A total of 89 non-repetitive MRSA isolates collected during routine veterinary microbiological examinations from April 2004 to the end of 2012, and one isolate from 2013 were used for this study. The presence of mecA and other resistance genes was confirmed by PCR. Isolates were genotyped by spa typing, two multiple-locus variable-number tandem repeat analyses (MLVA) analyses, SCCmec typing and multilocus sequence typing (MLST). PCR targeting Panton-Valentine leukocidin (PVL) and detection of staphylococcal enterotoxins (SE), toxic shock syndrome toxin (TSST) was performed using PCR assays. Antimicrobial susceptibility testing was performed.Five sequence types (STs—ST398, ST254, ST22, ST5 and ST1), SCCmec types II, IVa, V, and non-type-abele, 8 spa-types (t003, t011, t036, t127, t386, t1348, and t4450), and two isolates could not be assigned, 21 MLVA-14Orsay types Multiplex-PCR MLVA (mMLVA) displayed 17 different MLVA types.The present study is the most comprehensive dealing with MRSA from Austrian companion animals and horses. The results confirm that MRSA ST398 is present in a wide range of animal species and is predominant especially in horses. In other companion animals it is unclear whether the infections with the different MRSA isolates investigated in the present study truly represents a rare phenomenon or may be an emerging problem in companion animals.

Comparison of the epidemiological behavior of mastitis pathogens by applying time-series analysis in results of milk samples submitted for microbiological examination

The objective of this study is to examine and compare the trends of mastitis pathogens in quarter milk samples (n = 240,232) submitted for microbiological examination at the Milk Analysis Laboratory (L.I.G.A.L.) at Galicia, Spain from June 2005 to September 2011. Autoregressive Integrated Moving Average (ARIMA) models and multivariate statistical techniques such as Cluster Analysis were used in order to detect seasonal trends and similarities between the series trends and to classify mastitis pathogens into relatively homogeneous groups. The decrease of bulk milk somatic cell counts achieved by the mastitis control program, developed in recent years in this region, is the result of the decrease in IMI caused by a limited number of mastitis pathogens. The obtained results reflect a greater complexity in the behavior of mastitis pathogens, unlike the traditional classification into contagious or environmental. Staphylococcus aureus showed a trend similar to Streptococcus dysgalactiae, a mastitis pathogen can behave in both a contagious and an environmental manner. Among the traditionally considered environmental mastitis pathogens, Strep. uberis showed a different behavior to Escherichia coli and Klebsiella pneumoniae. Coagulase-negative staphylococci (CNS) species and Streptococcus other than Strep. agalactiae showed differences in the trend model. Time-series analysis and multivariate statistical techniques, such as Cluster Analysis, could be powerful tools to assess the isolation trend of mastitis pathogens because of their ability to cope with stochastic dependence of consecutive data. Furthermore, they could be used to identify the epidemiological behavior of mastitis pathogens using the results of milk samples submitted for routine microbiological examination, by classifying them into relatively homogeneous groups.

Actinobaculum schaalii, a commensal of the urogenital area

WHAT'S KNOWN ON THE SUBJECT? AND WHAT DOES THE STUDY ADD?: Actinobaculum schaalii is considered to be a part of the normai flora in the genital and urinary tract area. It has been associated to urinary tract infection (UTI), sepsis, osteomyelitis, endocarditis and Foumier's gangrene. So far it has mainly been isolated from urine, blood and pus, and predominantly in elderly patients. This study examined the habitat of A. schaalii by collecting samples from skin and urine in patients with kidney or ureter stones before and after treatment with Extracorporeal Shock Wave Lithotripsy (ESWL). Additionally faeces and vaginal swabs from routine specimen in patients not undergoing ESWL and without known urinary calculi were also analysed. The study does not find A. schaalii in faeces but shows it to be presents on skin and mucosa in the genital area. A. schaalii is also shown a possible pathogen in the stone-patient group undergoing ESWL. To study the habitat of Actinobaculum schaalii by examing groin swabs, faeces samples and vaginal swabs, and to determine whether it is a common uropathogen in patients with kidney or ureter stones. A quantitative real-time PCR assay was used to analyse all samples, which were collected between 2010 and 2011. A total of 38 patients (24 men and 14 women), with kidney or ureter stones and undergoing extracorporeal shock wave lithotripsy (ESWL), provided urine samples and had groin swabs taken. In addition, 30 faecal samples and 19 vaginal swabs that had been sent for routine microbiological examinations from patients outside the ESWL group were analysed. A chi-squared test was used to analyse the differences between patient groups, studying samples from urine, faeces samples, groin swabs and vaginal swabs. Actinobaculum schaalii was found in the urine samples from 14 (37%) patients undergoing ESWL, and in both urine and groin swabs from seven (18%) patients. Actinobaculum schaalii was not found in faeces samples but it was found in six (32%) of the vaginal swabs, predominantly in patients >50 years (P = 0.06). The study indicates that A. schaalii is a commensal found on skin, urine and vaginal mucosa in the human urogenital area and supports other investigations in its finding that the elderly are at greatest risk of being colonized with A. schaalii.

Vittaforma corneae keratitis in southern India: role of a novel duplex PCR

Microsporidia are obligate intracellular parasites that infect eukaryotic cells and have emerged as major opportunistic human pathogens. Due to the difficulties in definitive laboratory diagnosis and insufficient knowledge, ocular microsporidiosis is infrequently reported in India. To improve diagnostic facilities, we have developed a novel duplex PCR (dPCR) for the simultaneous identification of both genera and species of isolates with microsporidian aetiology that cause keratitis. The material scraped from the corneas of 12 clinically diagnosed microsporidial keratitis patients was subjected to routine microbiological examinations and molecular diagnosis using a novel dPCR that targeted the small-subunit rRNA gene (SSU-rRNA) of microsporidia and Vittaforma corneae using genus- and species-specific primers. Of the 12 corneal scrapes, 6 showed positive results in smears, while dPCR provided positive amplification with both pan-microsporidial and V. corneae species-specific primers for 9 corneal scrapes. The results were validated by sequencing and blast analysis. The sensitivity of this novel dPCR method was higher than that of conventional microscopy in the diagnosis of corneal microsporidial infection. dPCR with specific primers is potentially more sensitive, specific and depends less on more complicated methods for exact identification of the aetiology of microsporidial keratitis.

Bacteriological profile of corneal ulcer with references to Antibiotic susceptibility in a tertiary care hospital in West Bengal

Over one year, 80 OPD patients with corneal ulcers were scraped from the margins & base of the anaesthetized cornea & smear prepared for Gram staining & 10% KOH preparation. Blood agar for both aerobic & anaerobic, Mac-Conkey Agar, Chocolate Agar were inoculated. Turbidity in brain heart infusion were identified by Gram Stain & subsequently sub-cultured in Mac-Conkey & blood agar. Anaerobic jar containing the blood agar plates were incubated at 37 o C & examined after 48-72 hrs, & finally for 5 days & examined on alternative days before discarding. Further microbiological identification done as per standard protocol. All the isolated bacteria tested against different antimicrobial agents by standard disc diffusion method in accordance with CLSI guideline 2012. Out of 80 cases, corneal ulcer showed male preponderance (3:1) the highest no.19 (23.75%) cases were in the age group of 51-60 years. A total of 40 positive cultures were aerobes.12 specimens for anaerobic culture showed no growth. Among the isolates 19 (46.34%) were Staphylococcus aureus, 13 (31.7%) were CONS, 5 (12.19%) were Pseudomonas, 3 (7.31%) were E. Coli & 1 (2.43%) was Klebsiella. Gram positive cocci were maximally sensitive to Vancomycin, Tobramycin with highest resistance to Ciprofloxacin (55.87% sensitive). Gram negative isolates were maximally sensitive to Chloramphenicol & Moxifloxacin & resistant to Norfloxacin (44.44% sensitive). Routine microbiological examination of corneal ulcer is necessary to analyse & compare the changing trends in the microbial etiology & their susceptibility pattern to formulate a proper & appropriate antibiotic response against corneal ulcer.