Extended spectrum beta-lactamase and metallo beta-lactamase production among <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> isolated from different clinical samples in a tertiary care hospital in Kathmandu,Nepal

Background

Extended spectrum beta-lactamase (ESBL) and metallo beta-lactamase (MBL) production in Klebsiella pneumoniae and Escherichia coli are the commonest modes of drug resistance among these commonly isolated bacteria from clinical specimens. So the main purpose of our study was to determine the burden of ESBL and MBL production in E. coli and K. pneumoniae isolated from clinical samples. Further, the antimicrobial susceptibility patterns of E. coli and K. pneumoniae were also determined.

Methods

A cross-sectional study was conducted at Om Hospital and Research Centre, Kathmandu, Nepal by using the E. coli and K. pneumoniae isolated from different clinical samples (urine, pus, body fluids, sputum, blood) from May 2015 to December 2015. Antimicrobial susceptibility testing was performed by Kirby-Bauer disc diffusion technique. Extended spectrum beta-lactamase production was detected by combined disc method using ceftazidime and ceftazidime/clavulanic acid discs and cefotaxime and cefotaxime/clavulanic acid discs. Similarly, metallo beta-lactamase production was detected by combined disc assay using imipenem and imipenem/ethylenediaminetetracetate discs. Bacteria showing resistance to at least three different classes of antibiotics were considered multidrug resistant (MDR).

Results

Of total 1568 different clinical samples processed, 268 (17.1%) samples were culture positive. Among which, E. coli and K. pneumoniae were isolated from 138 (51.5%) and 39 (14.6%) samples respectively. Of the total isolates 61 (34.5%) were ESBL producers and 7 (4%) isolates were found to be MBL producers. High rates of ESBL production (35.9%) was noted among the clinical isolates from outpatients, however no MBL producing strains were isolated from outpatients. Among 138 E. coli and 39 K. pneumoniae, 73 (52.9%) E. coli and 23 (59%) K. pneumoniae were multidrug resistant. The lowest rates of resistance was seen toward imipenem followed by piperacillin/tazobactam, amikacin and cefoperazone/sulbactam.

Conclusions

High rate of ESBL production was found in the E. coli and K. pneumoniae isolated from outpatients suggesting the dissemination of ESBL producing isolates in community. This is very serious issue and can’t be neglected. Regular monitoring of rates of ESBL and MBL production along with multidrug resistance among clinical isolates is very necessary.

     

Molecular testing of <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> contaminating tissue allografts recovered from deceased donors

Microbiological screening of tissue allografts is crucial to prevent the transmission of bacterial and fungal infections to transplant recipients. Klebsiella was the most prevalent and resistant contaminating microorganism observed in our setting in the Iranian Tissue Bank. This study was conducted to determine the presence of extended-spectrum β-lactamase (ESBL) genes, antimicrobial resistance patterns of Klebsiella pneumoniae isolates, and their clonal relationships in allograft materials. K. pneumoniae contaminating bone and other tissue allografts recovered from deceased donors were identified and ESBL isolates were detected using a phenotypic confirmatory method. Antimicrobial susceptibility testing was carried out using the disk diffusion method. Distribution of ESBL genes and molecular typing were performed using polymerase chain reaction (PCR) and Repetitive-element (rep-PCR) methods. Of 3828 donated tissues, 51 (1.3%) were found contaminated by K. pneumoniae isolates. Compared to tissue allografts from brain-dead, heart-beating tissue donors, allografts from donors with circulatory cessation were associated with a higher risk of K. pneumoniae contamination [odds ratio (OR), 1.2 (CI 95% 0.9–2.3) (P value < 0.001)]. Half of the isolates produced ESBL, and the rate of susceptibility to cephalosporins was 51%. Among isolates, 22 (43.1%) harbored CTX-M, 31 (60.8%) SHV, and 9 (17.6%) harbored TEM types. The rep-dendrogram indicated that clones having identical or related strains with a similar antibiotype were isolated in the same period. This study provides evidence that a single clone of K. pneumoniae contaminated tissue allografts recovered from many different donors. A single clone found on tissues from several donors suggests contamination of tissues from a single source such as the tissue recovery process and environment. Genomic DNA testing and clonality of contaminating bacteria using molecular methods can focus the epidemiologic investigation on the tissue allograft recovery process including a search for contamination of the tissue recovery room environment, recovery staff, recovery equipment, reagents, solutions and supplies.     

Development of a Multiplex-PCR probe system for the proper identification of <Emphasis Type="Italic">Klebsiella variicola</Emphasis>

Background

Klebsiella variicola was very recently described as a new bacterial species and is very closely related to Klebsiella pneumoniae; in fact, K. variicola isolates were first identified as K. pneumoniae. Therefore, it might be the case that some isolates, which were initially classified as K. pneumoniae, are actually K. variicola. The aim of this study was to devise a multiplex-PCR probe that can differentiate isolates from these sister species.

Result

This work describes the development of a multiplex-PCR method to identify K. variicola. This development was based on sequencing a K. variicola clinical isolate (801) and comparing it to other K. variicola and K. pneumoniae genomes. The phylogenetic analysis showed that K. variicola isolates form a monophyletic group that is well differentiated from K. pneumoniae. Notably, the isolate K. pneumoniae 342 and K. pneumoniae KP5-1 might have been misclassified because in our analysis, both clustered with K. variicola isolates rather than with K. pneumoniae. The multiplex-PCR (M-PCR-1 to 3) probe system could identify K. variicola with high accuracy using the shared unique genes of K. variicola and K. pneumoniae genomes, respectively. M-PCR-1 was used to assay a collection of multidrug-resistant (503) and antimicrobial-sensitive (557) K. pneumoniae clinical isolates. We found K. variicola with a prevalence of 2.1% (23/1,060), of them a 56.5% (13/23) of the isolates were multidrug resistant, and 43.5% (10/23) of the isolates were antimicrobial sensitive. The phylogenetic analysis of rpoB of K. variicola-positive isolates identified by multiplex-PCR support the correct identification and differentiation of K. variicola from K. pneumoniae clinical isolates.

Conclusions

This multiplex-PCR provides the means to reliably identify and genotype K. variicola. This tool could be very helpful for clinical, epidemiological, and population genetics studies of this species. A low but significant prevalence of K. variicola isolates was found, implying that misclassification had occurred previously. We believe that our multiplex-PCR assay could be of paramount importance to understand the population dynamics of K. variicola in both clinical and environmental settings.

     

Spreading and mechanisms of antimicrobial resistance in microorganisms,producing beta-lactamases. Molecular mechanisms of resistance to beta-lactam antibiotics of <Emphasis Type="Italic">Klebsiella</Emphasis> spp. strains,isolated in cases of nosocomial infections

Antibiotic sensitivity has been investigated in nosocomial bacterial Klebsiella spp. strains isolated from patients treated in 30 hospitals of 15 Russian regions. Among Klebsiella strains (n = 212) studied the following species were found: Klebsiella pneumoniae ss. pneumoniae—182 (85.8%), Klebsiella pneumoniae ss. ozaenae—1 (0.5%), Klebsiella oxytoca—29 (13.7%) strains. Their sensitivity to antibacterial preparations was estimated by the method of serial dilutions in microvolume (the microdilution method). Carbapenems (imipenem and meropenem) exhibited the highest antibacterial activity against the strains studied. Among third generation cephalosporins the lowest MIC (Minimum inhibitory concentration) were found in the inhibitor protected preparations: ceftazidime/clavulanic acid (MIC₅₀ of 0.25 μg/ml; MIC₉₀ of 64 μg/ml) and cefoperazone/sulbactam (MIC₅₀ of 16 μg/ml; MIC₉₀ of 64 μg/ml). Using the PCR method the detection of class A betalactamases genes (TEM, SHV, CTX) was carried out in 42 strains of Klebsiella pneumoniae ss. pneumoniae. TEM type beta-lactamases were found alone or in various combinations in 16 (38.1%) strains, SHV—in 29 (69%), and CTX—in 27 (64.3%). Combinations of 2 and 3 different resistance determinants were detected in 23.8 and 26.2% of strains, respectively. Screening of carbapenem-resistant Klebsiella strains for production of class B metallo-beta-lactamases did not reveal nosocomial strains with phenotypically documented production of these enzymes.     

Antagonistic Activity of Lactic Acid Bacteria <Emphasis Type="Italic">Lactobacillus</Emphasis> spp. against Clinical Isolates of <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

The screening of three strains of lactic acid bacteria identified as Lactobacillus rhamnosus, Lactobacillus reuteri, and Lactobacillus helveticus showed significant antagonistic activity against Klebsiella pneumoniae strains characterized by multiple antibiotic resistance. Lactobacilli cocultivated with the Klebsiella strains inhibited their growth 20 to 86% on the first and second days, respectively. Exoproteome analysis of L. rhamnosus cocultivated with K. pneumoniae revealed the induction of peptidoglycan hydrolases, including extracellular lytic transglycosylases, family II (MltA), and endopeptidases capable of disrupting the peptidoglycan bacterial cell wall.     

Bacteriocin production by members of the genusKlebsiella

Bacteriocin production was tested in 36Klebsiella and 3Enterobacter aerogenes strains. Bacteriocins produced byK. pneumoniae were found to be active on most strains ofK. edwardsi, K. aerogenes, K. rhinoscleromatis andE. aerogenes. The bacteriocin produced byE. aerogenes 37 is also active onK. pneumoniae andK. ozaenae. The bacteriocins produced byK. rhinoscleromatis, K. edwardsi andK. aerogenes are active on only a few strains. The activity spectra of the bacteriocins of a number of strains were similar. The method of classification used for colicins could not be applied to these bacteriocins as mutants resistant to one bacteriocin were nearly always resistant to all other bacteriocins. One mutant, though resistant, still adsorbed the bacteriocin to which it was resistant and it is very likely that the same applies for all other resistant mutants. The hypothesis is made that allKlebsiella bacteriocins have the same biochemical target, or more likely, possess a common transmission mechanism.     

<Emphasis Type="Italic">Aeromonas</Emphasis> and <Emphasis Type="Italic">Plesiomonas</Emphasis> species from scarlet ibis (<Emphasis Type="Italic">Eudocimus ruber</Emphasis>) and their environment: monitoring antimicrobial susceptibility and virulence

The present study aimed at evaluating the role of captive scarlet ibises (Eudocimus ruber) and their environment as reservoirs of Aeromonas spp. and Plesiomonas spp., and analyzing the in vitro antimicrobial susceptibility and virulence of the recovered bacterial isolates. Thus, non-lactose and weak-lactose fermenting, oxidase positive Gram-negative bacilli were recovered from cloacal samples (n = 30) of scarlet ibises kept in a conservational facility and from water samples (n = 30) from their environment. Then, the antimicrobial susceptibility, hemolytic activity and biofilm production of the recovered Aeromonas spp. and Plesiomonas shigelloides strains were assessed. In addition, the virulence-associated genes of Aeromonas spp. were detected. Ten Aeromonas veronii bv. sobria, 2 Aeromonas hydrophila complex and 10 P. shigelloides were recovered. Intermediate susceptibility to piperacillin-tazobactam and cefepime was observed in 2 Aeromonas spp. and 1 P. shigelloides, respectively, and resistance to gentamicin was observed in 4 P. shigelloides. The automated susceptibility analysis revealed resistance to piperacillin-tazobactam and meropenem among Aeromonas spp. and intermediate susceptibility to gentamicin among P. shigelloides. All Aeromonas isolates presented hemolytic activity, while 3 P. shigelloides were non-hemolytic. All Aeromonas spp. and 3/10 P. shigelloides were biofilm-producers, at 28 °C, while 10 Aeromonas spp. and 6/10 P. shigelloides produced biofilms, at 37 °C. The most prevalent virulence genes of Aeromonas spp. were asa1 and ascV. Scarlet ibises and their environment harbour potentially pathogenic bacteria, thus requiring monitoring and measures to prevent contamination of humans and other animals.     

Genotypic and metabolic approaches towards the segregation of <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> strains producing different antibiotic resistant enzymes

Introduction

Genotype and metabolomic variation are important for bacterial survival and adaptation to environmental changes.

Objectives

In this study, we compared the relationship among Klebsiella pneumoniae strains based on their genotypic and metabolic profiles. In addition, we also evaluated the association of the relationship with beta-lactamase production.

Methods

A total of 53 K. pneumoniae strains isolated in 2013–2014 from a tertiary teaching hospital in Malaysia were subjected to antimicrobial susceptibility testing (AST) via disk diffusion method and beta-lactamase production confirmation. The bacterial strains were also typed genotypically and metabolically via REP-PCR and ¹H-NMR spectroscopy respectively. The concordance of the matrices derived based on genotypic and metabolic characterization was measured based on Spearman’s rank correlation.

Results

Spearman’s correlation rank showed that there is a weak but significant negative correlation between the genetic fingerprints and metabolic profiles of K. pneumoniae. Specifically, K. pneumoniae strains were clustered into five major clusters based on REP-PCR where most of the carbapenem resistant K. pneumoniae (CRKP) strains made up the major cluster. In contrast, metabolic patterns of the three groups (i.e. CRKP, extended spectrum beta-lactamase producing K. pneumoniae (ESBL), susceptible) of K. pneumoniae were clearly differentiated on PLS-DA score plots derived from ¹H-NMR spectroscopy.

Conclusion

Overall, this study showed that metabolomic profiling using ¹H-NMR spectroscopy is able to discriminate K. pneumoniae strains based on their beta-lactamase production status.

     

Comparison of virulence between matt and mucoid colonies of <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> coproducing NDM-1 and OXA-232 isolated from a single patient

Nine Klebsiella pneumoniae isolates coproducing NDM-1 and OXA-232 carbapenemases were successively isolated from a single patient. Although they were isolated simultaneously and were isogenic, they presented different colony phenotypes (matt and mucoid). All nine isolates were resistant to most antibiotics except colistin and fosfomycin. In addition, matt-type isolates were resistant to tigecycline. No differences were detected in the cps cluster sequences, except for the insertion of IS5 in the wzb gene of two matt-type isolates. In vitro virulence assays based on production of capsular polysaccharide, biofilm formation, and resistance to human serum indicated that the mucoid-type isolates were significantly more virulent than the matt-type. In addition, mucoid-type isolates showed higher survival rates than the matt-type ones in infection experiments in the fruit fly, suggesting a higher virulence of K. pneumoniae isolates with a mucoid phenotype. To our knowledge, this is the first report of K. pneumoniae colonies with different phenotypes being isolated from the same sample. In addition, we show that virulence varies with colony phenotype. Dissemination of K. pneumoniae isolates expressing both antibiotic resistance and high virulence would constitute a great threat.     

Biofilm and metallo beta-lactamase production among the strains of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> and <Emphasis Type="Italic">Acinetobacter</Emphasis> spp. at a Tertiary Care Hospital in Kathmandu,Nepal

Introduction

Pseudomonas aeruginosa and Acinetobacter spp. are found to be associated with biofilm and metallo-β-lactamase production and are the common causes of serious infections mainly in hospitalized patients. So, the main aims of this study were to determine the rates of biofilm production and metallo beta-lactamase production (MBL) among the strains of Pseudomonas aeruginosa and Acinetobacter spp. isolated from hospitalized patients.

Methods

A total of 85 P. aeruginosa isolates and 50 Acinetobacter spp. isolates isolated from different clinical specimens from patients admitted to Shree Birendra Hospital, Kathmandu, Nepal from July 2013 to May 2014 were included in this study. The bacterial isolates were identified with the help of biochemical tests. Modified Kirby-Bauer disc diffusion technique was used for antimicrobial susceptibility testing. Combined disc diffusion technique was used for the detection of MBL production, while Congo red agar method and tube adherence method were used for detection of biofilm production.

Results

Around 16.4% of P. aeruginosa isolates and 22% of the strains of Acinetobacter spp. were metallo β-lactamase producers. Out of 85 P. aeruginosa isolates, 23 (27.05%) were biofilm producers according to tube adherence test while, only 13 (15.29%) were biofilm producers as per Congo red agar method. Similarly, out of 50 Acinetobacter spp. 7 (14%) isolates were biofilm producers on the basis of tube adherence test, while only 5 (10%) were positive for biofilm production by Congo red agar method. Highest rates of susceptibility of P. aeruginosa as well as Acinetobacter spp. were seen toward colistin.

Conclusion

In our study, biofilm production and metallo beta-lactamase production were observed among Pseudomonas aeruginosa and Acinetobacter spp. However, no statistically significant association could be established between biofilm production and metallo beta-lactamase production.

     

Pig farm environment as a source of beta-lactamase or AmpC-producing <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> and <Emphasis Type="Italic">Escherichia coli</Emphasis>

The present study was undertaken to detect the occurrence of beta-lactamase-/AmpC-producing Klebsiella and Escherichia coli in healthy pigs, feed, drinking water, and pen floor or surface soil. The study also intended to detect the clonal relationship between the environmental and porcine isolates to confirm the route of transmission. Rectal swabs and environmental samples were collected from apparently healthy pigs kept in organized or backyard farms in India. The pigs had no history of antibiotic intake. Production of phenotypical beta-lactamase, associated genes, and class I integron gene was detected in E. coli and Klebsiella isolates. The phylogenetic relationship among the isolates was established on the basis of Random amplification of polymorphic DNA banding pattern. Beta-lactamase-producing Klebsiella were isolated from healthy pigs (20.0%), pen floor swabs/surface soil swabs (14.0%), and drinking water (100%). Escherichia coli isolated from healthy pigs (14.4%), pen floor/surface soil (8.0%), and drinking water (33.3%) were detected as beta-lactamase producers. Majority of beta-lactamase-producing isolates possessed bla_CTX-M-9. Further, 35 (81%) Klebsiella and all the E. coli isolates were detected as AmpC beta-lactamase ACBL producers and possessed bla_AmpC. Sixteen beta-lactamase-producing Klebsiella (37.20%) and 13 E. coli (86.67%) possessed class I integron. Few resistant isolates from environmental sources (surface soil swab and drinking water) and the studied pigs were detected within the same cluster of the dendrogram representing their similarities. The study indicated about the possible role of contaminated environment as a source of beta-lactamase/AmpC-producing Klebsiella and E. coli in pigs.     

Incidence of metallo-beta-lactamase-producing <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> isolates from hospital setting in Pakistan

The aim of this study was monitoring and surveillance in different wards of the PIMS hospital, Islamabad, to understand emerging challenges of antibiotic resistance in particular association with most virulent serotypes of Klebsiella pneumoniae. The study was conducted during March 2015 to September 2015. The study showed that rate of isolation of K. pneumoniae was 37% (103 positives out of a total of 277 clinical samples) and 7.7% (8) were phenotypically and genotypically confirmed to be metallo-β-lactamase resistant (carbapenem resistant) and all of them were multidrug resistant (MDR). These carbapenem-resistant isolates were isolated from blood, endotracheal tubes, and pus. Molecular screening for the presence of integrons indicated that distribution of class I integrons (87.5% of carbapenem-resistant K. pneumoniae isolates) was higher than class II integrons (1.25%) among given isolates. The study indicated that exposure of metallo-beta-lactamase-producing strains through hospitalizations increases the chances of spread of MDR pathogens. There is an urgent need for effective surveillance and monitoring strategies to control the spread of extremely resistant K. pneumoniae implicated in nosocomial infections leading to the increased health burden and enforcement of policy guideline on appropriate antibiotics usage.     

Colistin resistance in <Emphasis Type="Italic">Enterobacter</Emphasis> spp. isolates in Korea

We investigated the colistin resistance rate among 356 Enterobacter spp. clinical isolates from eight hospitals in Korea. Antibiotic susceptibility testing was performed by broth microdilution. While 51 of 213 (23.9%) Enterobacter cloacae isolates were colistin-resistant, only six of 143 (4.2%) E. aerogenes isolates showed resistance. We also identified the skip well phenotype in eight E. cloacae and three E. aerogenes isolates. Multilocus sequence typing for E. cloacae and randomly amplified polymorphic DNA analysis and enterobacterial repetitive intergenic consensus PCR for E. aerogenes revealed that clonal spreading of colistin-resistant and skip well Enterobacter spp. isolates had not occurred. In vitro time-kill assays were performed with three colistin-resistant, three skip well, and two colistin-susceptible isolates of E. cloacae and E. aerogenes. Inconsistent results were observed among isolates with skip well phenotypes; while some were eradicated by 2 mg/L colistin, others were not. This suggests that skip well isolates have differentiated into different categories. As the high rates of colistin resistance in E. cloacae detected are of clinical concern, continuous monitoring is warranted. In addition, the clinical implications and mechanisms of the skip well phenotype should be investigated to ensure the appropriate use of colistin against Enterobacter infections.     

Virulence-associated genes and antibiotic susceptibility among vaginal and rectal <Emphasis Type="Italic">Escherichia coli</Emphasis> isolates from healthy pregnant women in Poland

Vaginal and/or rectal Escherichia coli colonization of pregnant women is sometimes associated with neonatal infections. Despite the relevance of these strains, they have been rarely described before. Thus, the aim of this study was to compare vaginal (VEC) and rectal E. coli (REC) isolates in respect of antimicrobial susceptibility and the frequency of virulence-associated genes (VAGs). The antimicrobial susceptibility of 50 VEC and 50 REC isolates was performed by using the disc diffusion method, and VAGs were detected by PCR. There were no significant differences in the antimicrobial resistance between VEC and REC. Both VEC and REC isolates were mostly resistant to ticarcillin (36 and 30%) and ampicillin (36 and 22%). None of the tested isolates was positive for ESBL. Gene’s fimH, fimA, sfa/foc, iutA, ibeA, hlyF, and neuC were detected, respectively, in 98, 92, 32, 28, 12, 8, and 2% of VEC and in 94, 72, 12, 34, 8, 10, and 8% of REC isolates. The co-occurrence of fimA/H and sfa/foc genes was significantly more prevalent among VEC isolates, in comparison to REC isolates. The study indicated that VEC and REC isolates are quite similar in terms of antimicrobial non-susceptibility and VAGs.     

Assessment of antibiotic resistance in <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> exposed to sequential in vitro antibiotic treatments

Background

Bacteria treated with different classes of antibiotics exhibit changes in susceptibility to successive antibiotic treatments. This study was designed to evaluate the influence of sequential antibiotic treatments on the development of antibiotic resistance in Klebsiella pneumoniae associated with β-lactamase and efflux pump activities.

Methods

The antibiotic susceptibility, β-lactamase activity, and efflux activity were determined in K. pneumoniae grown at 37 °C by adding initial (0 h) and second antibiotics (8 or 12 h). Treatments include control (CON; no first and second antibiotic addition), no initial antibiotic addition followed by 1 MIC ciprofloxacin addition (CON-CIP), no initial antibiotic addition followed by 1 MIC meropenem addition (CON-MER), initial 1/4 MIC ciprofloxacin addition followed by no antibiotic addition (1/4CIP-CON), initial 1/4 MIC ciprofloxacin addition followed by 1 MIC ciprofloxacin addition (1/4CIP-CIP), and initial 1/4 MIC ciprofloxacin addition followed by 1 MIC meropenem addition (1/4CIP-MER).

Results

Compared to the CON, the initial addition of 1/4 MIC ciprofloxacin inhibited the growth of K. pneumoniae throughout the incubation period. The ciprofloxacin treatments (CON-CIP and 1/4CIP-CIP) showed significant reduction in the number of K. pneumoniae cells compared to meropenem (CON-MER and 1/4CIP-MER). The 1/4CIP-CIP achieved a further 1 log reduction of K. pneumoniae, when compared to the 1/4CIP-CON and 1/CIP-MER. The increase in sensitivity of K. pneumoniae to cefotaxime, kanamycin, levofloxacin, nalidixic acid was observed for CON-CIP. Noticeable cross-resistance pattern was observed at the 1/4CIP-CIP, showing the increased resistance of K. pneumoniae to chloramphenicol, ciprofloxacin, kanamycin, levofloxacin, nalidixic acid norfloxacin, sulphamethoxazole/trimethoprim, and tetracycline. The levels of β-lactamase activities were estimated to be 8.4 μmol/min/ml for CON, 7.7 μmol/min/ml for 1/4CIP-CON and as low as 2.9 μmol/min/ml for CON-CIP. Compared to the absence of phenylalanine-arginine-β-naphthylamide (PAβN), the fluorescence intensity of EtBr was increased in K. pneumoniae cells treated at the CON, CON-CIP, and CON-MER in the presence of PAβN. However, the efflux pump activity remained in K. pneumoniae cells treated at the 1/CIP, 1/CIP–CIP, and 1/CIP-MER in the presence of PAβN.

Conclusion

The results suggest that the pre-exposed antibiotic history, treatment order, and concentrations influenced the development of multiple antibiotic resistant associated with β-lactamase and efflux pump activities. This study highlights the importance of antibiotic treatment conditions, which would be taken into consideration when new antibiotic strategy is designed to prevent antibiotic resistance.

     

Characterization of <Emphasis Type="Italic">Streptococcus pneumoniae</Emphasis> isolates from Austrian companion animals and horses

Background

The aim of the present study was to investigate the genetic relatedness and the antimicrobial resistance profiles of a collection of Austrian Streptococcus pneumoniae isolates from companion animals and horses. A total of 12 non-repetitive isolates presumptively identified as S. pneumoniae were obtained during routinely diagnostic activities between March 2009 and January 2017.

Results

Isolates were confirmed as S. pneumoniae by bile solubility and optochin susceptibility testing, matrix-assisted laser desorption-ionization-time of flight (MALDI-TOF) mass spectrometry and sequence analysis of a part recA and the 16S rRNA genes. Isolates were further characterized by pneumolysin polymerase chain reaction (PCR) and genotyped by multilocus sequence typing (MLST). Antimicrobial susceptibility testing was performed and resistance genes were detected by specific PCR assays. All isolates were serotyped. Four sequence types (ST) (ST36, ST3546, ST6934 and ST6937) and four serotypes (3, 19A, 19F and 23F) were detected. Two isolates from twelve displayed a multidrug-resistance pheno- and genotype.

Conclusions

This study represents the first comprehensive investigation on characteristics of S. pneumoniae isolates recovered from Austrian companion animals and horses. The obtained results indicate that common human sero- (23F) and sequence type (ST36) implicated in causing invasive pneumococcal disease (IPD) may circulate in dogs. Isolates obtained from other examined animals seem to be host-adapted.

     

Antimicrobial misuse in pediatric urinary tract infections: recurrences and renal scarring

Background

In children, urinary tract infection (UTI) is one of a common bacterial infection. This study was conducted to detect the uropathogen, antimicrobial susceptibility, pathogen associated with recurrences and renal scarring in children initially taken care from general practitioners and later presented to tertiary care.

Methods

Every inward UTI episode, culture and antimicrobial susceptibility was done while on past 6-month, history of infections and use of antimicrobials was collected using clinical records and demonstration of antimicrobials. Children with recurrent pyelonephritis was followed and in vitro bio film formation was assessed.

Results

Frequency of UTI was significantly high among infants (p?=?0.03). Last 6-month, all (220) were exposed to antimicrobials. Cefixime was the commonly prescribed antimicrobial (p?=?0.02). In current UTI episode, 64.5% (142/220) of children with UTI were consulted GPs’ prior to seek treatment from tertiary care pediatric unit (p?=?0.02). While on follow up child who developed UTI, found urine culture isolates were significantly shifted from E. coli and K. pneumoniae to extended spectrum of beta-lactamase (ESBL) E. coli and K. pneumoniae. Out of 208 participants, 36 of them had re-current pyelonephritis (R-PN). Renal scarring (RS) was detected in 22 out of 70 patients with pyelonephritis following dimercaptosuccinic acid scan. Following each episodes of recurrent pyelonephritis 11% of new scar formation was detected (p?=?0.02). Bio film forming E. coli and K. pneumoniae was significantly associated in patients with R-PN (p?=?0.04).

Discussion

Medical care providers often prescribe antimicrobials without having an etiological diagnosis. While continuing exposure of third generation cephalosporin and carbapenem leads to development of ESBL and CRE microbes in great. The empiric uses of antimicrobials need to be stream lined with local epidemiology and antimicrobial susceptibility pattern. R-PN in childhood leads to RS. In great, bio film formation act as the focus for such recurrences.

     

Fecal Carriage of ESBL-Producing E. coli and K. pneumoniae in Children in Guinea-Bissau: A Hospital-Based Cross-Sectional Study

Background

In recent years, the world has seen a surge in extended-spectrum β-lactamase (ESBL)-producing bacteria. However, data on the dissemination of ESBL-producing Enterobacteriaceae in the community from systematically enrolled study subjects in Africa remains limited. To determine the prevalence, phenotypic resistance patterns and genetic characteristics of ESBL-producing E. coli and K. pneumoniae in fecal carriage and to analyze associated risk factors in children attending a pediatric emergency department in Guinea-Bissau.

Methodology/Principal Findings

From June to September 2010, children <5 years of age with fever or tachycardia attending a pediatric emergency ward during the day was screened for ESBL carriage in feces. Socio-demographic and health seeking behavior data was collected. Antibiotic susceptibility was tested with VITEK2 and EUCAST disk diffusion method, molecular characterization of ESBL-encoding genes was performed with multiplex PCR and clonal relatedness was established by automated rep-PCR. Of 408 enrolled children 133 (32.6%) were ESBL carriers. In total, 83 E. coli and 91 K. pneumoniae ESBL-producing isolates were obtained. Nearly all isolates were multidrug-resistant. Co-resistance to ciprofloxacin, trimethoprim-sulfamethoxazole and aminoglycosides was common. Of the isolates, 38.5% were co-resistant to these classes plus extended-spectrum cephalosporins, which infers resistance to all easily available antibiotic agents for treatment of gram-negative sepsis in Guinea-Bissau. The predominant resistance-encoding gene subgroup was bla _CTX-M-1 and epidemiologic typing showed that the bacterial ESBL population was highly diverse both for E. coli and K. pneumoniae. Bed sharing with another child <5 years of age was a risk factor for ESBL carriage, indicating crowding as a potential risk factor for transmission of ESBL-producing bacteria.

Conclusions/Significance

Prevalence of ESBL-producing bacteria in this population was high and clonally diverse. This is alarming considering the limited diagnostic and treatment possibilities in Guinea-Bissau and other resource-poor countries.     

In Vitro Susceptibility of a Large Collection of <Emphasis Type="Italic">Candida</Emphasis> Strains Against Fluconazole and Voriconazole by Using the CLSI Disk Diffusion Assay

We evaluated all Candida spp. isolates obtained from patients admitted to two tertiary care hospitals between 1999 and 2003 in the city of São Paulo, Brazil. The in vitro activities of fluconazole (FCZ) and voriconazole were determined by the agar disk diffusion test using the Clinical and Laboratory Standards Institute M44-A guidelines. The inhibition zone diameters were read and interpreted automatically by the BIOMIC^® image-analysis plate reader system. We tested a total of 4,625 strains, including 2,393 strains of C. albicans (51.7%), 658 of C. tropicalis (14.2%), 503 of C. glabrata (10.9%), 495 of C. parapsilosis (10.7%), 292 of C. rugosa (6.3%), 195 of C. guilliermondii (4.2%) and 89 of other Candida species (2.0%). Only 2.0% of the strains tested were classified as dose-dependent susceptible (DDS), and 5.8% of them were resistant to FCZ. The resistance or DDS to fluconazole was verified mainly among C. glabrata (7.8%), C. krusei (67.9%) and C. rugosa (65.1%). Voriconazole exhibited better activity in vitro than fluconazole, even in isolates fluconazole resistant. The resistance of fluconazole and voriconazole did not increase in the isolates of Candida spp. during the evaluated period.     

Incidence of Klebsiella Species in Surface Waters and Their Expression of Virulence Factors

To investigate the occurrence of different Klebsiella spp. in aquatic environments, a total of 208 samples of natural surface waters was examined. From half (53%) of these samples, 123 Klebsiella strains were isolated, the most common species being Klebsiella pneumoniae. A comparison of these isolates to a group of 207 clinical K. pneumoniae isolates demonstrated that water isolates of K. pneumoniae, unlike those of K. oxytoca and K. planticola, are as capable as clinical isolates of expressing putative virulence factors such as serum resistance and capsular polysaccharides, pili, and siderophores.