- Volume 68, Issue 3, 2019

Purpose. The effect of a combination of polymyxin B (PMB) and vancomycin (VAN) was assessed against six Acinetobacter baumannii clinical isolates belonging to six different clusters (three PMB-susceptible and three PMB-resistant).

Methodology. The synergistic effect of the PMB–VAN combination was determined with the checkerboard, time-kill, disk-diffusion and M.I.C.Evaluator assays. PMB-resistance was investigated with mcr-1 gene amplification and a mutant frequency assay.

Results. In the checkerboard assay, all PMB-resistant isolates showed a synergistic effect. The time-kill assay demonstrated that the PMB–VAN combination had a bactericidal effect at 24 h against isolates with a high mutant rate for PMB, suggesting that this combination may block the hypermutation of some isolates. No antagonism was detected. All PMB-resistant isolates also showed synergism in the disk-diffusion test, and a significant decrease in VAN MICs in the M.I.C.Evaluator assay.

Conclusion. Our findings indicate that the PMB–VAN combination has a synergistic effect on A. baumannii , especially against PMB-resistant isolates.

Purpose. The epidemiological shift in MRSA distribution from healthcare-related facilities to the general population is distressing and requires continuous monitoring to manage and control the rate of incidences.

Method. The retrospective relationship between genetic and phenotypic variability of methicillin-resistant Staphylococcus aureus (MRSA) isolates was determined in respect to the specimen source, patient location, sex and age. A total of 521 MRSA isolates were classified based on SCCmec, mec, agr, pvl and spa genetic markers using three different multiplex PCRs.

Results. Based on the genetic variability, the isolates were divided into 97 profiles, of which 59% belonged to only two profiles (P17 and P33). P17 was the predominate profile, harbouring SCCmecIVa, ccr2, mecB, agr1, spa413 and pvl markers. P17 was more prevalent among the younger population (average 33.9 years) from outpatient (77%) locations and wound (88%) sources. The second largest profile was P33, harbouring SCCmecII, ccr2+ccr3, mecA, agr2, spa413 and no PVL. P33 was more prevalent in the older population (average 70.7 years) and more common in females (62%) than males (38%). With respect to antibiotic resistance, P33 exhibited a high rate of resistance to penicillins, cephalosporins, fluoroquinolones and macrolides, and P17 had a lower resistance to fluoroquinolones.

Conclusion. This report contributes to the existing understanding of evolutionary epidemiology of antibiotic resistance in MRSA. The diversity of MRSA isolates and unique environmental preferences for each profile highlights the importance of epidemiological knowledge of MRSA distribution to determine the best treatment for patients in both community and hospital settings.

Purpose . To highlight the clinical significance of carbapenem-resistant Klebsiella pneumoniae (CRKP) rectal colonization by examining the risk factors for CRKP rectal colonization and subsequent bloodstream infection (BSI) in critically ill patients.

Methodology . Prospective study of CRKP rectal colonization in an intensive care unit (ICU) during a 39-month period. CRKP strains isolated from both the blood cultures and corresponding rectal specimens (n=96) of patients were screened by PCR for the presence of antibiotic resistance-associated genes. Molecular analyses were conducted to investigate the clonal relatedness of CRKP strains from the rectal and blood specimens.

Results . Among the 498 patients, 226 were rectally colonized by CRKP, 48 of whom developed a CRKP BSI. The median time from hospital admission to the detection of CRKP rectal colonization was 8 days, while the median time from colonization to BSI was 4 days. The duration of ICU stay, patient/nurse ratio and prior use of antianaerobic antimicrobials were associated with CRKP rectal colonization. No specific factor was associated with BSIs in the colonized patients. The bla _KPC-2 gene was detected in all 96 strains, which were all classified as sequence type ST-258. Representative pairs (n=48) of CRKP strains colonizing and infecting the same patient shared the same pulsotype.

Conclusion . Our results indicate that hospitalized patients become infected with their colonizing strains, supporting the strong association between colonization and BSI. Limiting antianaerobic antimicrobial administration, reducing the duration of ICU stay and maintaining a low patient/nurse ratio are possible strategies to restrict rectal CRKP colonization in ICUs.

A multidrug-resistant Pseudomonas aeruginosa PS1 isolated from urine clinical sample was investigated in this study. The strain exhibited resistance to piperacillin/tazobactam, ciprofloxacin, imipenem, ceftazidime but it was susceptible to colistin. Analysis of whole-genome sequencing data by ResFinder detected various resistance determinants including qnrVC1 and bla _NDM-1. The multiresistant P. aeruginosa isolate belonged to ST773 high-risk clone. The qnrVC1 and bla _NDM-1 determinants were incorporated into different integrons. Expression of bla _NDM-1 was fourfold and qnrVC1 was twofold increased, compared to that of rpsL housekeeping gene. Mutations in gyrA Thr83Leu and parC Ser87Leu were detected and additionally qnrVC1 expression indicates protective effect of QnrVC1 pentapeptid protein on gyrase and topoisomerase. High-risk P. aeruginosa clones integrate various carbapenemase and other resistance determinants into their genomes that facilitates further dissemination of multiresistance among clinical isolates. We report bla _NDM-1 and qnrVC1 genes in P. aeruginosa ST773 international high-risk clone.

Purpose . The prevalence of antimicrobial-resistant bacteria, especially cephalosporin-resistant Enterobacteriaceae , is a major concern for human and animal health. We investigated the prevalence of cephalosporin-resistant Enterobacteriaceae among sheltered dogs and cats with various backgrounds.

Method . Faecal samples or rectal swabs were collected from 151 dogs and 182 cats, and screened for the presence of antimicrobial-resistant bacteria. Isolates were characterized phenotypically and genotypically by pulsed-field gel electrophoresis, multi-locus sequence typing and phylogenetic grouping. The animal attributes related to bacterial carriage were statistically analysed.

Results . Cephalosporin-resistant Enterobacteriaceae was detected in 22 dogs (14.6%) and 20 cats (11.0%): 21 were extended-spectrum β-lactamase (ESBL)-producing, 20 were AmpC-producing, and 1 was both ESBL- and AmpC-producing. Their β-lactamase genes were varied and associated with humans, animals or other origins. The genes CTX-M-14 (n=9) and CMY-2 (n=9) were dominant, but CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-15, CTX-M-24, CTX-M-27, CTX-M-55 and DHA-1 genes were also detected. Genotyping of isolates revealed that β-lactamase-producing Enterobacteriaceae had high genetic diversity. Relationships between animals harbouring cephalosporin-resistant Enterobacteriaceae and individual attributes, such as sex and nutrition type, were detected, but there was no correlation between history of human association and the presence of the bacterium in either dogs or cats.

Conclusion . We found several types of cephalosporin-resistant Enterobacteriaceae distributed among companion animals with a range of individual attributes and histories in Osaka, Japan. Companion animals may play a bridging role in the circulation of antimicrobial-resistant bacteria from humans and from other origins.

Purpose. Non-albicans Candida species have emerged as fungal pathogens that cause invasive infections, with many of these species displaying resistance to commonly used antifungal agents. This study was confined to studying the characteristics of clinical isolates of the C. rugosa complex and C. pararugosa species.

Methodology. Seven isolates of the C. rugosa complex and one isolate of C. pararugosa were obtained from two tertiary referral hospitals in Malaysia. Their antifungal susceptibilities, biofilm, proteinase, phospholipase, esterase and haemolysin activities were characterized. Biofilms were quantified using crystal violet (CV) and tetrazolium (XTT) reduction assays at 1.5, 6, 18, 24, 48 and 72 h.

Results/Key findings. The E-test antifungal tests showed that both species have elevated MICs compared to C. albicans and C. tropicalis. The highest biomass was observed in one of the C. rugosa isolates (0.237), followed by C. pararugosa (0.206) at 18 h of incubation. However, the highest bioactivity was observed in the C. rugosa ATCC 10571 strain at 24 h (0.075), followed by C. pararugosa at 48 h (0.048) and the same C. rugosa strain at 24 h (0.046), with P<0.05. All isolates exhibited high proteinase activity (+++) whereas six isolates showed very strong esterase activity (++++). All the isolates were alpha haemolytic producers. None of the isolates exhibited phospholipase activity.

Conclusion. Elevated MICs were shown for the C. rugosa complex and C. pararugosa for commonly used antifungal drugs. Further studies to identify virulence genes involved in the pathogenesis and genes that confer reduced drug susceptibility in these species are proposed.

Purpose. The standard culture findings for detecting and identifying bacterial pathogens in patients with lower respiratory tract infections (LRTIs) are usually not available for two to three days, which delays the initiation of appropriate antibiotic therapies. We aimed to develop a faster method of identification of bacterial pathogens in LRTIs which would offer a timelier guide to initial antibiotic choices.

Methodology. The developed PCR-pyrosequencing-based method was defined as mask PCR-pyrosequencing (MPP). This method uses primer pairs deliberately designed to mask the interference of colonised bacteria in sputum to detect and identify bacterial pathogens directly from LRTI patient sputum samples within 5 h. Accordingly, the standard PCR-pyrosequencing method was defined as normal PCR-pyrosequencing (NPP) here. The clinical performance of the novel system was evaluated by comparing with traditional semi-quantitative culture and identification results.

Results. The coincidence for culture and MPP was 91.3 %. Compared with the semi-quantitative culture results, NPP identified 89.9 % strains of grade 3+ (corresponding to 1.0×106 c.f.u ml−1) and 100 % of grade 4+ (corresponding to 1.0×107 c.f.u ml−1), both of which were considered to be the presumptive pathogens in the clinics. MPP identified 98.9 % strains of grade 3+ and 100 % of grade 4+. Additionally, PCR-pyrosequencing could detect a minimum concentration of 1.0×106 c.f.u ml−1 of bacteria in sputum, with no significant difference between NPP and MPP.

Conclusion. The PCR-pyrosequencing technique developed in this study is an accurate, fast, and high throughput method for the direct detection and identification of bacterial pathogens from sputum.

Purpose . Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) has become a considerable public health concern in both developed and developing countries due to the rapid spread of this bacterium around the world, also the epidemiology of MRSA has changed, as the isolation of MRSA strains is not limited to health-care settings or patients with predisposing risk factors. Therefore, the objective of this study is to determine the genetic diversity and antibiotic resistance profile of CA-MRSA nasal carriage in Iranian children.

Methodology . A cross-sectional study was conducted from April 2013 to March 2014. A total of 25 CA-MRSA were isolated from the anterior nares of 410 preschool children with no risk factors. All MRSA isolates were characterized by detection of the Panton–Valentine leukocidin (pvl) and γ-hemolysin genes, staphylococcal cassette chromosome mec (SCCmec) typing and multi-locus sequence typing (MLST).

Results . In 25 CA-MRSA isolates, Pvl and γ-hemolysin genes were detected in one (4%) and 18 (72 %) isolates; respectively. Overall, 92% (23/25) of isolates belonged to SCCmec type IV and 8% (2/25) of them had SCCmec type V profile. Using MLST, the 25 isolates were grouped into six clonal complexes (CC) and eight sequence types (ST) (CC5/ST6, CC22/ST22 and ST217, CC30/ST30 and ST1107, CC78/ST859, CC398/ST291 and CC97/ST405). The ST859/SCCmec IV (11/25, 44%) was the predominant clone among the isolates. ST859-MRSA-IV-pvl-negative (resistant to tetracycline) have successfully adapted to the Iranian preschool children population.

Conclusion . Our results suggest that the genomic diversity was observed among the CA-MRSA. In addition, the current study demonstrates that pvl is not a reliable marker for CA-MRSA in our region.

Purpose. The global spread and increasing clinical impact of carbapenemase-producing bacteria are alarming. Rapid diagnostic techniques for carbapenemase detection are of the utmost importance to prevent delays in efficient antibiotic therapy and the control of spread in hospitals. Recently, multiplex immunochromatographic lateral flow tests (ICTs) for the fast detection of carbapenemase-producers have become commercially available. We evaluated a novel multiplex ICT for the rapid detection of OXA-48, KPC, NDM and VIM carbapenemases.

Methodology. One hundred well-characterized multidrug-resistant Enterobacteriaceae were analysed by RESIST-4 (Coris, BioConcept, Gembloux, Belgium). The reference standard included confirmation at the molecular level at the German National Reference Laboratory for multidrug-resistant Gram-negative bacteria (Ruhr-University Bochum, Germany).

Results/Key findings. The tested ICT was highly reliable, showing 100 % sensitivity and 100 % specificity.

Conclusion. As it is fast, easy to perform and has few technical requirements, the ICT represents a good opportunity to improve turnaround times and patient care for the clinical microbiology laboratory.

Purpose . To analyse the role of virulence factors (VFs) and host in Klebsiella pneumoniae upper urinary tract infections (UTIs) in renal transplant (RTx) recipients.

Methodology. Clinical and demographic data were registered prospectively. Phylogenetic background of K. pneumoniae isolates was analysed by PCR melting profiles (MP) and the following VFs genes: fimH-1, uge, kpn, ycfM, mrkD, rmpA, magA, hlyA, cnf-1, irp-1, irp-2, fyuA, entB, iutA, iroN by PCR.

Results. We studied urine cultures and clinical data from 61 episodes of K. pneumoniae UTI in 54 RTx recipients. There were 32 cases of AB (53%), 10 cases of lower UTI (16%), 19 cases of AGPN (31%), including six cases of bacteraemia. In total, 74 % of strains were extended-spectrum beta-lactamase+, and there were two carbapenemase-producing strains. PCR MP typing showed a diverse population with 52 different genetic profiles of K. pneumoniae . Analysis of the DNA profiles indicated 45 unrelated, unique genotypes and 7 related (16 isolates from 15 patients) genotypes. Urine flow impairment emerged as an independent predictor of K. pneumoniae upper UTIs (OR 14.28, CI 2.7–75.56, P 0.002), while we did not find any association between the profile of VFs and developing upper UTIs. The prevalence of the uge gene was lower in RTx patients on everolimus when compared to isolates from patients not receiving mTOR inhibitors (33.3 % vs 82.8 % P<0.05).

Conclusions . K. pneumoniae upper UTI may be a marker of urine flow impairment. Bacterial VFs could not discriminate between upper and lower UTIs. However, immunosuppression may influence the selection of particular VFs.

Purpose. Lowenstein–Jensen (LJ) medium used for cultivating Mycobacterium tuberculosis (MTB) is marketed in glass packaging. Breakage of glass slope is a major biosafety risk, especially during processing and storage, which gets magnified in large laboratories. We evaluated the performance of new bioMérieux (bMx) LJ slopes in plastic packaging, compared to bMx glass LJ medium and Becton Dickinson Mycobacterial Growth Indicator Tube (MGIT), for MTB recovery.

Methodology. Consecutive pulmonary/extra-pulmonary samples (n=240) were processed using routine methods of decontamination, inoculation and incubation.

Results. Plastic LJ slopes detected all 213 true-positive cases. The mean time-to-growth detection was 17.97 days for plastic LJ slopes, compared to 18.08 and 13.53 days for glass LJ slopes and MGIT, respectively. No statistically significant difference was observed between the two LJ slopes (P< 0.05). Both LJ slopes had a sensitivity and specificity of 100%, with respect to MGIT.

Conclusion. Plastic LJ slopes are a good alternative to the traditional glass slopes. The medium quality did not differ with the packaging material. Increased surface area of these slants allowed enhanced growth, and the clear plastic material allowed accurate recording of growth. The wide mouth of these containers eased inoculation. Increased biosafety, by elimination of breakage risk, is the biggest advantage of this modification.

Purpose. To address more information about changes in commensal Escherichia coli during virus intestinal infection, we characterized 30 faecal E. coli isolates from calves (21 to 60 days old) with diarrhea due to rotavirus and coronavirus, which received, before diagnosis, tetracycline, gentamicin and enrofloxacin drugs.

Methodology. Clermont’s phylogenetic classification; presence of genes for curli, cellulose, fimbriae (F4, F5, F6, F18, F41); and antimicrobial susceptibility were used to characterize the isolates. Disk diffusion technique and PCR were used as methodologies.

Results. E. coli isolates from calves with diarrhea were phylogenetically classified as B1 (70%, 21/30), B2 (3.33%, 1/30), C (3.33%, 1/30), D (3.33%, 1/30), E (13.33%, 4/30) and unknown (6.7 %; 2/30), whereas E. coli isolates from the control group were classified only as B1 (83.3%, 25/30), E (10 %; 3/30) and unknown (6,7 %; 2/30). E. coli isolates from calves with diarrhea showed a much higher resistance profile with 16 (53.3%) multiresistant isolates. Only isolates (30%-9/30) from diarrheic calves were also positive for fimbriae, specifically 16.7% (5/30) for F5 and 13.3% (4/30) for F18.

Conclusion. To sum up, E. coli isolates from calves with diarrhea showed differences in relation to the control group, confirming changes in commensal E. coli during virus intestinal infection. It can be emphasized that some care should be taken to manage diarrheic calves: the pathological agent must be diagnosed prior to treatment; antibacterial treatment should be with antimicrobials with a different mechanism of action; and finally, treated animals should be maintained separately from others because they can carry micro-organisms with a resistant profile.