Pulsed-field Gel Electrophoresis Analysis Research Articles

Emergence and Characterization of a Novel IncP-6 Plasmid Harboring bla KPC-2 and qnrS2 Genes in Aeromonas taiwanensis Isolates.

The dissemination of Klebsiella pneumoniae carbapenemases (KPCs) among Gram-negative bacteria is an important threat to global health. However, KPC-producing bacteria from environmental samples are rarely reported. This study aimed to elucidate the underlying resistance mechanisms of three carbapenem-resistant Aeromonas taiwanensis isolates recovered from river sediment samples. Pulsed-field gel electrophoresis (PFGE) and whole genome sequencing (WGS) analysis indicated a close evolutionary relationship among A. taiwanensis isolates. S1-PFGE, Southern blot and conjugation assays confirmed the presence of blaKPC–2 and qnrS2 genes on a non-conjugative plasmid in these isolates. Plasmid analysis further showed that pKPC-1713 is an IncP-6 plasmid with a length of 53,205 bp, which can be transformed into DH5α strain and mediated carbapenems and quinolones resistance. The plasmid backbone of p1713-KPC demonstrated 99% sequence identity to that of IncP-6-type plasmid pKPC-cd17 from Aeromonas spp. and IncP-6-type plasmid: 1 from Citrobacter freundii at 74% coverage. A 14,808 bp insertion sequence was observed between merT gene and hypothetical protein in p1713-KPC, which include the quinolone resistance qnrS2 gene. Emergence of plasmid-borned blaKPC and qnrS2 genes from A. taiwanensis isolates highlights their possible dissemination into the environment. Therefore, potential detection of such plasmids from clinical isolates should be closely monitored.

Determination of antibiotic susceptibility, ESBL genes and pulsed-field gel electrophoresis profiles of extended-spectrum β-lactamase-containing Escherichia coli isolates

The purpose of this study was to determine the phenotypic antibiotic susceptibility patterns, extended-spectrum β-lactamase (ESBL) genes and genotypic profiles of ESBL-positive Escherichia coli strains isolated from urine samples obtained from outpatients with urinary tract infection in Turkey. A total of 120 E. coli strains during 2017, 2018, and 2019 (40 patients per year) were examined for antibiotic susceptibility patterns by disc diffusion method, for ESBL genes using PCR and sequencing and for molecular typing by pulsed-field gel electrophoresis (PFGE) method. The isolates were evaluated for their sensitivity to 21 different antibiotics. Four different antimicrobial resistance patterns were determined according to antibiotic susceptibility status of the isolates. The β-lactamase genes detected in the isolates were CTX-M-15 + OXA-1 (n= 14), CTX-M-15 (n= 24), TEM-1 + CTX-M-15 (n= 52), TEM-1 + SHV-12 (n=6), SHV-12 1 (n= 6), TEM-1 + CTX-M-1 (n= 6), TEM-1 + CTX-M-16 (n= 6) and TEM-1 + CTX-M-9 (n= 6). The CTX-M-15 was the most prevalent ESBL enzyme in the isolates. As a result of PFGE analysis performed by XbaI enzyme restriction process, one major PFGE profile and three main groups (Group I-II-III) were observed. While antibiotic resistance profiles of the strains showed four groups (RI-RII-RIII-RIV), PFGE band profiles showed a major group (90% similarity ratio). High ESBL production and decreased susceptibility to broad-spectrum cephalosporins were observed in E. coli strains. In addition, PFGE analysis showed high clonal similarity among E. coli isolates.

Rapid identification of Klebsiella pneumoniae isolates from various samples with biosensor and genotyping

The most important cause of Klebsiella spp. contamination of drinking water is the leakage of animal faeces into drinking water sources. Recently, the biosensor technology has quickly begun to replace other methods with its faster finding and reliability. The aim of this study was to investigate the reliability of the biosensor technology in the rapid detection of Klebsiella pneumoniae (K. pneumoniae) and to determine the presence of the relationship between K. pneumoniae isolates isolated from the drinking water thought to be contaminated by animal faeces and the clinical isolates. For this purpose, portable, microfluidic electrochemical sensor device version 2 (V2) was used for the detection of K. pneumoniae and results were confirmed with VITEK MALDI-TOF Mass Spectrometry (VITEK MS) automated system. For the molecular typing of K. pneumoniae isolates, pulsed-field gel electrophoresis (PFGE) and multiple locus variable-number tandem repeat analysis (MLVA) methods were employed and the results were compared. For these bacteria, the most appropriate typing method was tried to be determined comparatively. PFGE analysis indicated the presence of six different strains, while MLVA divided them into 23 clusters. Clonal relationships were viewed between environmental and clinical isolates. The main goal of this paper is to present, the detailed report of the comparison of the samples isolated from drinking water, animal and human faeces for K. pneumoniae. To accomplish of this goal we introduced that MLVA and PFGE methods. Also, gold nanoparticies enhanced electrochemical biosensor device is used for the determination of K. pneumoniae for the first time.

Antibiogram and molecular characterization of methicillin-resistant Staphylococcus aureus recovered from treated wastewater effluent and receiving surface water in Durban, South Africa.

Municipal wastewater treatment plants (WWTPs) may serve as a reservoir for potentially pathogenic and antibiotic resistant bacteria. The discharge of improperly treated wastewater effluent may lead to the spread of these bacteria, including methicillin-resistant Staphylococcus aureus (MRSA) which is responsible for causing pneumonia, septicaemia and skin and soft tissue infections, into the receiving surface waters. This study aimed to determine the antibiogram and virulence gene profiles of MRSA isolates recovered from treated wastewater effluent and receiving surface waters. Genetic fingerprinting of the isolates was also carried out to determine the phylogenetic relationship between the isolates and selected antibiogram profiles. Eighty MRSA isolates were obtained from treated effluent and receiving rivers of two WWTPs in Durban, KwaZulu-Natal. Antibiotic resistance was observed towards lincomycin (100%), oxacillin (98.75%), cefoxitin and penicillin (97.50%), and ampicillin (96.25%). In addition, 72.50%, 66.25%, 52.50%, 40% and 33.75% of isolates showed resistance against cefozolin, azithromycin, amoxicillin/clavulanic acid, erythromycin and vancomycin, respectively. Antibiotic resistance genes detected in the isolates tested in this study: aac(6')/aph(2″) (56.25%), ermC (62.50%), msrA (22.50%), and blaZ and tetK (70%). The frequency of virulence genes: hla and sea was 57.50%, hld was 1.25%, while lukS P/V was 0%. Pulse Field Gel Electrophoresis analysis generated 13 pulsotypes (designated A-M) showing a correlation to their respective antibiograms. Findings from this study showed the presence of potentially pathogenic, multi-drug resistant MRSA in the treated effluent and receiving surface waters. This may have detrimental effects on the health of individuals who come into contact with these water resources.

Emergence of two Escherichia coli strains co-harboring mcr-1 and bla NDM in fresh vegetables from China.

BackgroundThe concurrence of mcr and carbapenemase genes among Enterobacteriaceae has been a great clinical concern. In our study, we aimed to investigate the prevalence of mcr-positive carbapenem-resistant Enterobacteriaceae (CRE) in fresh vegetables and shed light on the possibility of transmission of mcr-positive CRE via fresh vegetables.MethodsIn this study, 712 fresh vegetable samples from 10 provinces in China were collected between May 2017 and Dec 2018 and were screened for mcr and carbapenemase genes. Antibiotic susceptibilities for isolates co-harboring carbapenemase genes and mcr were determined by an agar dilution or a broth microdilution method. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) analysis were also performed. Transferability of the carbapenemase/mcr-bearing plasmids was determined by conjugation, replicon typing and S1-PFGE-Southern blotting. The sequences of these plasmids were analyzed by using whole-genome sequencing with Illumina Hiseq platform.ResultsTwo E. coli isolates concomitantly carrying mcr-1 and blaNDM-5/9 from leaf rape and spinach, respectively, were found and both isolates showed multidrug resistance. Notably, mcr-1-positive 690 harboring blaNDM-5 and 701 carrying blaNDM-9 belonged to ST156 and ST2847, respectively, similar to the prevalent MLST types of E. coli co-carrying mcr-1 and blaNDM from avian in our previous study. mcr-1 was on ~33-kb IncX4 plasmid or ~60-kb IncI2 plasmid, while blaNDM-5/9 was on ~46-kb IncX3 plasmid or ~120-kb untypable plasmid. The plasmids were highly similar to those from animals and clinical patients reported in various countries.Conclusion: E. coli isolates concomitantly carrying mcr-1 and blaNDM-5/9 in fresh vegetables may serve as a direct source of pathogens in humans, and such discovery in fresh vegetables emphasizes the importance of prompt surveillance and intervention in limiting the spread of E. coli co-carrying blaNDM and mcr-1. To our knowledge, this is the first report of Enterobacteriaceae co-carrying blaNDM and mcr-1 in fresh vegetables.

An outbreak of severe infectious diseases caused by methicillin-resistant Staphylococcus aureus USA300 clone among hospitalized patients and nursing staff in a tertiary care university hospital

The USA300 clone, which produces Panton-Valentine leukocidin (PVL), is a major highly pathogenic community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) clone that is spreading throughout the world. Although the prevalence of the USA300 clone in Japan was very limited a decade ago, its incidence has been increasing in both community and hospital settings in recent years. There is great concern that the USA300 clone will cause more complicated diseases and become a serious threat to immunocompromised patients in hospital settings. Here, we report an outbreak of severe infectious diseases in a tertiary care university hospital involving the incidence of deep infections, including bacteremia, and continuous and frequent isolation of MRSA strains for five months from six patients and a healthy nursing staff member in the same ward. The genotype of all MRSA isolates was identical to that of the USA300 clone. Furthermore, pulsed-field gel electrophoresis analysis indicated that all MRSA had the same patterns. These data demonstrate that a USA300 clone outbreak had occurred in the hospital. Fortunately, this outbreak was terminated subsequent to the interventions of the infection control team and all patients recovered following the appropriate therapies. Our report demonstrates that patients carrying highly pathogenic CA-MRSA have the potential to become a source of nosocomial outbreaks that can spread to healthy healthcare workers. Therefore, stricter standard precautions should be applied for all patients at the time of admission to prevent such nosocomial outbreaks.

Molecular Epidemiology of Multidrug-Resistant Klebsiella pneumoniae Isolates in a Brazilian Tertiary Hospital.

Multidrug-resistant (MDR) Klebsiella pneumoniae (Kp) is a major bacterial pathogen responsible for hospital outbreaks worldwide, mainly via the spread of high-risk clones and epidemic resistance plasmids. In this study, we evaluated the molecular epidemiology and β-lactam resistance mechanisms of MDR-Kp strains isolated in a Brazilian academic care hospital. We used whole-genome sequencing to study drug resistance mechanisms and their relationships with a K. pneumoniae carbapenemase-producing (KPC) Kp outbreak. Forty-three Kp strains were collected between 2003 and 2012. Antimicrobial susceptibility testing was performed for 15 antimicrobial agents, and polymerase chain reaction (PCR) was used to detect 32 resistance genes. Mutations in ompk35, ompk36, and ompk37 were evaluated by PCR and DNA sequencing. Pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were carried out to differentiate the strains. Based on distinct epidemiological periods, six Kp strains were subjected to whole-genome sequencing. β-lactamase coding genes were widely distributed among isolates. Almost all isolates had mutations in porin genes, particularly ompk35. The presence of blaKPC promoted a very high increase in carbapenem minimum inhibitory concentration only when ompk35 and ompk36 were interrupted by insertion sequences. A major cluster was identified by PFGE analysis and all isolates from this cluster belonged to clonal group (CG) 258. We have also identified a large repertoire of resistance genes in the sequenced isolates. A blaKPC–2-bearing plasmid (pUFPRA2) was also identified, which was very similar to a plasmid previously described in the first Brazilian KPC-Kp (2005). We found high-risk clones (CG258) and an epidemic resistance plasmid throughout the duration of the study (2003 to 2012), emphasizing a persistent presence of MDR-Kp strains in the hospital setting. Finally, we found that horizontal transfer of resistance genes between clones may have played a key role in the evolution of the outbreak.

Prevalence and characterization of multidrug resistance and variant Salmonella genomic island 1 in Salmonella isolates from cattle, poultry and humans in Iran.

Salmonella enterica is a common food-borne pathogen with occasional multidrug resistance (MDR). Salmonella genomic island (SGI1) is a horizontally transmissible genomic island, containing an MDR gene cluster. All Salmonella serotypes are public health concern, although there is an additional concern associated with those that harbour SGI1. In Iran, there are no data on the presence of SGI1 variants in Salmonella isolates. The present study was conducted to identify MDR- and SGI1-carrying Salmonella strains isolated from various sources and to compare their genetic relatedness between human and animal sources. In total, 242 Salmonella isolates collected from chicken, cattle, and humans from 2008 through 2014 were studied. The isolates were tested for resistance to 14 antimicrobials via the disc diffusion method. They were also tested for the presence of SGI1 variants via PCR, and genetic relatedness was evaluated based on pulsed-field gel electrophoresis (PFGE). Resistance to at least one antimicrobial agent was observed in 132 (54%) Salmonella isolates (n=242), while more than 40% of the isolates showed MDR. Based on PCR analysis, eight variants of SGI1, including SGI1, SGI1-B, SGI1-C, SGI1-D, SGI1-F, SGI1-I, SGI1-J and SGI1-O, were found in both human and animal isolates. Statistical analysis revealed no significant difference in the prevalence of SGI1 variants between human and animal isolates (p>0.05). Macrorestriction PFGE analysis of the isolates with the same SGI1 variant and resistance patterns revealed genetic relatedness ranging from 70% to 100% among human and animal isolates. According to our review, this is the first documentation of SGI1 in Salmonella isolates in Iran. The presence of similar SGI1 variants in both humans and animals, along with their related PFGE patterns, suggests that food-producing animals may be a source of MDR Salmonella isolates in Iran.

Non-biogroup 1 or 2 Strains of the Emerging Zoonotic Pathogen Escherichia albertii, Their Proposed Assignment to Biogroup 3, and Their Commonly Detected Characteristics.

Escherichia albertii, a zoonotic enteropathogen, is responsible for outbreaks of disease in humans. Identifying strains of E. albertii by phenotypic characterization tests is difficult because of its poorly defined properties. Screening its phenotypic characteristics is, nevertheless, a necessary prerequisite for further genetic analysis of its properties, and species-specific polymerase chain reaction (PCR) analysis can be used to type the pathogen. While two E. albertii biogroups (1 and 2) have been described, strains with characteristics divergent from both biogroups have been reported worldwide. The aim of the present study was to evaluate the characteristics of non-biogroup 1 or 2 strains, and discern the characteristics common to all of the E. albertii strains from this study. Altogether, 107/414 field isolates were selected for examination based on pulsed-field gel electrophoresis analysis. The 107 strains were isolated from 92 sources, including humans and pigeon feces, other wild birds, and retail chicken livers. All strains were then examined using various culture-based, biochemical (API 50CHE tests, API Zym test, and others) and molecular (virulence gene screening, multi-locus sequence analysis) testing methods. Our results revealed that all field strains (n = 107) showed non-biogroup 1 or 2 characteristics, with multiple sequence differences. Variations in indole production and the lysine decarboxylase activity profiles among the isolates made identification of E. albertii very difficult. Therefore, we propose that non-biogroup 1 or 2 of E. albertii should be assigned to biogroup 3 to make screening of them easier in public health and clinical laboratory settings. Clearly, having group criteria for indole-negative/lysine-positive, indole-positive/lysine-negative, and indole-positive/lysine-positive E. albertii biogroups 1, 2, and 3 strains, respectively, should provide for more accurate identification of E. albertii isolates. Based on our findings, we recommend that isolates displaying phenotype mobility-negativity (sulfide-indole-motility medium, 37°C), hydrogen sulfide production-negativity (triple sugar iron medium), acid production-negativity from xylose, negative β-glucuronidase activity properties, and showing indole production and lysine decarboxylase activity profiles in accordance with one of the three biogroups, should be further assessed using an E. albertii-specific PCR assay.

Frequency and expression of genes involved in adhesion and biofilm formation in Staphylococcus aureus strains isolated from periodontal lesions.

The aim of this study was to characterize the Staphylococcus aureus strains isolated from periodontal lesions of patients, to determine the expression of genes involved in cell adhesion upon their infection of human epithelial cells using an invitro model, its biofilm formation, and its resistance to antibiotics. S. aureus was analysed by PCR, Kirby-Bauer, and pulsed-field gel electrophoresis (PFGE), measuring gene expression by real-time PCR after infection of human cells invitro. S. aureus was identified in 18.6% (50/268) of the samples. All strains (n=50) possessed the virulence genes spa (Staphylococcal protein A), coa (coagulase), and icaAB (intercellular adhesin); 96% (n=48) possessed clfB (clumping factor B), and 88% (n=44) possessed ebps (elastin-binding protein) and sdrD (serine aspartate repeat protein D). All strains were resistant to methicillin, ampicillin, dicloxacillin, cefotaxime, and penicillin, and were multidrug resistant to 6-12 antibiotics. PFGE analysis showed 37 different pulsed-field types and most strains (60.4%) had a unique pulsed-field type. Twenty-four distinct combinations of virulence genes and antibiotic-resistant phenotypes were identified. Although S. aureus has been considered a transient member of the oral microbiota, our results indicate a high-level expression of virulence genes and multidrug resistance in the strains isolated from periodontal lesions. These strains might complicate the successful treatment of the disease.

A prospective multicenter study on the evaluation of antimicrobial resistance and molecular epidemiology of multidrug-resistant Acinetobacter baumannii infections in intensive care units with clinical and environmental features

BackgroundMultidrug-resistant (MDR) Acinetobacter baumannii infections are considered as emerging nosocomial infections particularly in patients hospitalized in intensive care units (ICUs). Therefore, reliable detection of MDR strains is crucial for management of treatment but also for epidemiological data collections. The purpose of this study was to compare antimicrobial resistance and the clonal distribution of MDR clinical and environmental A. baumannii isolates obtained from the ICUs of 10 different hospitals from five geographical regions of Turkey in the context of the demographic and clinical characteristics of the patients.MethodsA multicenter-prospective study was conducted in 10 medical centers of Turkey over a 6 month period. A total of 164 clinical and 12 environmental MDR A. baumannii isolates were included in the study. Antimicrobial susceptibility testing was performed for amikacin (AN), ampicillin–sulbactam (SAM), ceftazidime (CAZ), ciprofloxacin (CIP), imipenem (IMP) and colistin (COL) by microdilution method and by antibiotic gradient test for tigecycline (TIG). Pulsed-field gel electrophoresis (PFGE) was performed to determine the clonal relationship between the isolates. The detection of the resistance genes, blaOXA-23, blaOXA-24, blaOXA-51,blaOXA-58,blaIMP,blaNDM, blaKPC, blaOXA-48 and blaPER-1 was carried out using the PCR method.ResultsThe mortality rate of the 164 patients was 58.5%. The risk factors for mortality included diabetes mellitus, liv1er failure, the use of chemotherapy and previous use of quinolones. Antimicrobial resistance rates for AN, SAM, CAZ, CIP, IMP, COL and TIG were 91.8%, 99.4%, 99.4%, 100%, 99.4%, 1.2% and 1.7% respectively. Colistin showed the highest susceptibility rate. Four isolates did not grow on the culture and were excluded from the analyses. Of 172 isolates, 166 (96.5%) carried blaOXA-23, 5 (2.9%) blaOXA-58 and one isolate (0.6%) was positive for both genes. The frequency of blaPER-1 was found to be 2.9%. None of the isolates had blaIMP, blaKPC, blaNDM and blaOXA-48 genes. PFGE analysis showed 88 pulsotypes. Fifteen isolates were clonally unrelated. One hundred fifty-seven (91.2%) of the isolates were involved in 14 different clusters.ConclusionsColistin is still the most effective antibiotic for A. baumannii infections. The gene blaOXA-23 has become the most prevalent carbapenemase in Turkey. The distribution of invasive A. baumannii isolates from different regions of Turkey is not diverse so, infection control measures at medical centers should be revised to decrease the MDR A. baumannii infections across the country. The results of this study are expected to provide an important baseline to assess the future prophylactic and therapeutic options.

PulseNet Latin America and the Caribbean Network: Present and Future.

PulseNet Latin America and Caribbean (PNLAC) was established in 2003 and is one of seven Regional networks within PulseNet International. The main objectives of the network are to strengthen national and regional laboratory-based foodborne disease surveillance for early detection and investigation of outbreaks to setup control and prevention strategies in contribution to Public Health. Participants perform standardized pulsed-field gel electrophoresis (PFGE) protocols and analysis. For functioning, it is important for the network, the development of national and regional databases (RDBs) and the communication between countries, regionally and internationally. Metadata from over 8600 cases/outbreaks are profiled and isolated by PFGE and are incorporated into the RDB, hosted by the Pan American Health Organization. Currently PNLAC is moving toward whole-genome sequencing to use as a complementary strategy for surveillance. The aim of this article was to describe the experience of the construction of PNLAC, and its contribution to the surveillance of the foodborne diseases at the country and regional levels.

Emerging erm(B)-Mediated Macrolide Resistance Associated with Novel Multidrug Resistance Genomic Islands in Campylobacter.

The rapid dissemination of the macrolide resistance gene erm(B) will likely compromise the efficacy of macrolides as the treatment of choice for campylobacteriosis. More importantly, erm(B) is always associated with several multidrug resistance genomic islands (MDRGIs), which confer resistance to multiple other antimicrobials. Continuous monitoring of the emergence of erm(B) and analysis of its associated genetic environments are crucial for our understanding of macrolide resistance in Campylobacter In this study, 290 Campylobacter isolates (216 Campylobacter coli isolates and 74 Campylobacter jejuni isolates) were obtained from 1,039 fecal samples collected in 2016 from pigs and chickens from three regions of China (344 samples from Guangdong, 335 samples from Shanghai, and 360 samples from Shandong). Overall, 74 isolates (72 C. coli isolates and 2 C. jejuni isolates) were PCR positive for erm(B). Combined with data from previous years, we observed a trend of increasing prevalence of erm(B) in C. coli Pulsed-field gel electrophoresis analyses suggested that both clonal expansion and horizontal transmission were involved in the dissemination of erm(B) in C. coli, and three novel types of erm(B)-associated MDRGIs were identified among the isolates. Furthermore, 2 erm(B)-harboring C. jejuni isolates also contained an aminoglycoside resistance genomic island and a multidrug-resistance-enhancing efflux pump, encoded by RE-cmeABC Antimicrobial susceptibility testing showed that most of the isolates were resistant to all clinically important antimicrobial agents used for the treatment of campylobacteriosis. These findings suggest that the increasing prevalence of erm(B)-associated MDRGIs might further limit treatment options for campylobacteriosis.

Weaned piglets: another factor to be considered for the control of Salmonella infection in breeding pig farms

Field studies on Salmonella infection in suckling piglets are scarce due to the intrinsic difficulties of collecting proper samples (i.e. tonsils or mesenteric lymph nodes), and most of them rely on the analysis of rectal swabs that limit their accuracy. We used 495 slaughtered 4-weeks-old male piglets intended for human consumption from 5 Salmonella-seropositive breeding farms to collect gastrointestinal packages and perform a thorough detection of Salmonella on mesenteric lymph nodes and intestinal content. The overall prevalence of both infection and shedding was high (≈ 36%) indicating that piglets played an active role in Salmonella maintenance in the farms. Major serotypes found in piglets included 4,[5],12:i: (35.4%), Rissen (17.1%), Derby (10.9%) and Bovismorbificans (10.3%). In most of the infected animals (72.8%) the same serotype was found in mesenteric lymph nodes and feces. Significant higher ELISA OD% values were found in meat juice samples from non-infected piglets compared to infected ones (median OD% of 12.0 and 17.3, respectively; P = 0.002) suggesting some protective effect of sow’s colostrum. Salmonella was also isolated from feces from weaned sows contemporary of the slaughtered piglets, and 89% of the serotypes identified in sows were also detected in piglets. Pulsed field gel electrophoresis analyses showed that 75% of the piglet isolates that were compared to those of sows were related to them, suggesting the circulation of Salmonella strains between sows and piglets. It appears that improving piglet colostrum intake along with the reduction of the shedding in sows may favor the control of Salmonella infection in breeding farms.

Population Dynamics of Thermotolerant Campylobacter in Broilers Reared on Reused Litter.

A study using sentinel broiler chickens was performed to address Campylobacter persistence in litter that was reused for successive flocks. Cloacal swabs, litter, drag swabs, darkling beetles, feed, and drinking water were weekly sampled and analyzed by standard microbiological procedures. Thermotolerant Campylobacter isolated strains were confirmed by polymerase chain reaction and subtyped by pulsed-field gel electrophoresis analysis. Campylobacter was not detected in samples collected immediately after downtime between broiler flocks. However, Campylobacter-positive samples were first detected at 21 d. After Campylobacter was initially isolated from the cloacal swabs, reused litter, drag swabs, or darkling beetles, these samples remained Campylobacter positive in the following weeks until the end of the rearing period. Campylobacter-positive cloacal swabs obtained from sentinel broilers ranged from 97.3% to 100% at 42 d. All isolated strains were identified as Campylobacter jejuni. Among the subtypes identified, an indistinguishable C. jejuni strain was predominant in sentinel broilers and was also detected in the other environmental samples analyzed, suggesting a common and persistent contamination source within the flocks. Sentinel broilers may have contributed to amplify the Campylobacter level, maintaining flock and broiler house contamination until the end of the production cycle.

Uropathogenic Escherichia coli strains harboring tosA gene were associated to high virulence genes and a multidrug-resistant profile

TosA, a putative repeats-in-toxin protein that has recently gained importance as an antigenic molecule, has characteristics of nonfimbrial adhesins and can act as a virulence marker in uropathogenic Escherichia coli (UPEC) strains; however, little is known about the association of this protein with antibiotic resistance profiles in UPEC tosA+ clinical strains. The aim of this study was to evaluate UPEC tosA+ strains, including examining genetic diversity, associations with phylogenetic groups, resistance profiles, virulence genes, adherence assays, integrons, and extended-spectrum beta-lactamase phenotypes. Pulsed-field gel electrophoresis analysis grouped these strains into eight clusters with 62% genetic diversity. These strains were mainly associated with the multidrug-resistant profiles, together with an association with class 1 integron and the extended-spectrum beta-lactamase phenotype. Additionally, the strains exhibited a distribution of ≥96% for core-associated genes, while a variable distribution was identified for pathogenic islands-associated genes. Strong associations between UPEC tosA+ strains and two phylogenetic groups (B2 and D) were identified, including resistance to β-lactam and non-β-lactam antibiotics. The UPEC tosA+ clinical strains exhibited major adherence, which was related to the fitness and virulence genes. A recombinant TosA protein reacted with antibodies from the sera of urinary tract infection patients, and anti-recombinant TosA polyclonal antibodies also detected TosA expression in these strains. In conclusion, strains of UPEC tosA+ belonging to phylogenetic group B2 had a high frequency of fitness and virulence genes associated with class 1 integrons and the extended-spectrum beta-lactamase phenotype, which exhibited a high adherence profile. The TosA protein is expressed during infection with UPEC and is considered an immunogenic molecule.

Whole Genome Sequencing Based Surveillance of L. monocytogenes for Early Detection and Investigations of Listeriosis Outbreaks.

In Austria, all laboratories are legally obligated to forward human and food/environmental L. monocytogenes isolates to the National Reference Laboratory/Center (NRL) for Listeria. Two invasive human isolates of L. monocytogenes serotype 1/2a of the same pulsed-field gel electrophoresis (PFGE) pattern, previously unknown in Austria, were cultured for the first time in January 2016. Five further human isolates, obtained from patients with invasive listeriosis between April 2016 and September 2017, showed this PFGE pattern. In Austria the NRL started to use whole-genome sequencing (WGS) based typing in 2016, using a core genome MLST (cgMLST) scheme developed by Ruppitsch et al. 2015, which contains 1701 target genes. Sequence data are submitted to a publicly available nomenclature server (Ridom GmbH, Münster, Germany) for allocation of the core genome complex type (CT). The seven invasive human isolates differed from each other with zero to two alleles and were allocated to CT1234 (declared as outbreak strain). Among the Austrian strain collection of about 6,000 cgMLST-characterized non-human isolates (i.e., food/environmental isolates) 90 isolates shared CT1234. Out of these, 83 isolates were traced back to one meat processing-company. They differed from the outbreak strain by up to seven alleles; one isolate originated from the company's industrial slicer. The remaining seven CT1234-isolates were obtained from food products of four other companies (five fish-products, one ready-to-eat dumpling and one deer-meat) and differed from the outbreak strain by six to eleven alleles. The outbreak described shows the considerable potential of WGS to identify the source of a listeriosis outbreak. Compared to PFGE analysis, WGS-based typing has higher discriminatory power, yields better data accuracy, and allows higher laboratory through-put at lower cost. Utilization of WGS-based typing results of human and food/ environmental L. monocytogenes isolates by appropriate public health analysts and epidemiologists is indispensable to support a successful outbreak investigation.

Hidden Carbapenem Resistance in OXA-48 and Extended-Spectrum β-Lactamase-Positive Escherichia coli.

The purpose of this study was to report the identification OXA-48 carbapenemase in seven extended-spectrum β-lactamase (ESBL)-positive Escherichia coli clinical isolates, fully susceptible to all carbapenems by disk diffusion and E-test methods, but with borderline minimal inhibitory concentration (MIC) values of ertapenem. This report points to the necessity for determination of carbapenem MICs in ESBL-positive E. coli isolates and additional phenotypic testing for carbapenemases in all isolates with borderline ertapenem MIC defined by EUCAST. The isolates showed a high level of resistance to expanded-spectrum cephalosporins because of the production of an additional ESBL belonging to CTX-M family. All isolates and their respective tranconjugants were found to possess L plasmid. Pulsed-field gel electrophoresis analysis revealed two clusters containing highly related isolates. The global spread of multidrug-resistant E. coli should be monitored closely because of the ability of isolates to rapidly obtain additional antibiotic resistance traits such as plasmid-mediated OXA-48 genes.

Overestimation of an Outbreak of Enterobacter cloacae in a Neonatal Intensive Care Unit in Germany, 2015.

In August 2015, 17 neonates with Enterobacter cloacae (E. cloacae) colonization were identified in a neonatal intensive care unit (NICU) in Germany. Two developed severe brain abscesses. Despite temporary NICU closure in September, another infant with E. cloacae colonization was detected in October 2015. We defined potential cases as inpatients treated in the NICU or any pediatric/maternity ward in 2015 with E. cloacae in any specimen before molecular typing. Cases were at first confirmed by arbitrarily-primed-polymerase-chain-reaction and later by XbaI-macrorestriction/pulsed-field gel electrophoresis and next-generation-sequencing. Enhanced barrier precautions and cohorting were implemented for all potential cases and microbiologic screening was extended from NICU to all pediatric/maternity wards. Of 41 potential cases (occurring between 08/04/2015 and 15/11/2015 in 4 wards), the isolates of 23 shared identical arbitrarily-primed-polymerase-chain-reaction patterns; 3 without plausible epidemiologic link. Pulsed-field gel electrophoresis analyses verified only 10 cases (all in the NICU); next-generation-sequencing analysis confirmed these results. In addition 6 cases without isolates available for genotyping were closely linked in place and time. Forty-one suspected patients were cohorted and the NICU was temporarily closed. Further analyses revealed that only 16 cases belonged to the outbreak. Only close interdisciplinary collaboration and highly discriminatory genotyping methods allowed to clearly differentiate between cases and noncases in this E. cloacae outbreak.

Laboratory characterisation of Salmonella enterica serotype Typhi isolates from Zimbabwe, 2009\u20132017

BackgroundTyphoid fever remains a major public health problem in Zimbabwe with recurrent outbreaks reported since 2009. To provide guidance on appropriate treatment choice in order to minimise the morbidity and mortality of typhoid fever and prevent large scale outbreaks, we investigated the antimicrobial susceptibility patterns, prevalence of Salmonella enterica serotype Typhi (S. Typhi) H58 haplotype and molecular subtypes of S. Typhi from outbreak strains isolated from 2009 to 2017 in Zimbabwe and compared these to isolates from neighbouring African countries.MethodsAntimicrobial susceptibility testing was performed on all isolates using the disk diffusion, and E-Test, and results were interpreted using Clinical and Laboratory Standards Institute (CLSI) guidelines (2017). S. Typhi H58 haplotype screening was performed on 161 (58.3%) isolates. Pulsed-field gel electrophoresis (PFGE) was performed on 91 selected isolates across timelines using antibiotic susceptibility results and geographical distribution (2009 to 2016).ResultsBetween 2009 and 2017, 16,398 suspected cases and 550 confirmed cases of typhoid fever were notified in Zimbabwe. A total of 276 (44.6%) of the culture-confirmed S. Typhi isolates were analysed and 243 isolates (88.0%) were resistant to two or more first line drugs (ciprofloxacin, ampicillin and chloramphenicol) for typhoid. The most common resistance was to ampicillin-chloramphenicol (172 isolates; 62.3%). Increasing ciprofloxacin resistance was observed from 2012 to 2017 (4.2 to 22.0%). Out of 161 screened isolates, 150 (93.2%) were haplotype H58. Twelve PFGE patterns were observed among the 91 isolates analysed, suggesting some diversity exists among strains circulating in Zimbabwe. PFGE analysis of 2013, 2014 and 2016 isolates revealed a common strain with an indistinguishable PFGE pattern (100% similarity) and indistinguishable from PFGE patterns previously identified in strains isolated from South Africa, Zambia and Tanzania.ConclusionsResistance to first line antimicrobials used for typhoid fever is emerging in Zimbabwe and the multidrug resistant S. Typhi H58 haplotype is widespread. A predominant PFGE clone circulating in Zimbabwe, South Africa, Zambia and Tanzania, argues for cross-border cooperation in the control of this disease.