A Community Waterborne Salmonella Bovismorbificans Outbreak in Greece.

BACKGROUND: In August 2022, the Hellenic National Public Health Organisation was notified about a gastroenteritis outbreak in town A in Southern Greece. Investigations aimed to identify the source and implement control measures. METHODS: Case definition categories were used in a 1:3 case-control study. Cases and controls were interviewed about various exposures. Cases' stool samples were cultured on agar plates and characterised by serotyping, antimicrobial susceptibility testing and Pulse Field Gel Electrophoresis (PFGE). Environmental investigations included tap water sampling for microbiological and chemical analysis in town A and inspection of the water supply system. RESULTS: We identified 33 cases (median age: 17 years). Tap water consumption was the only significant risk factor for gastroenteritis (OR = 5.46, 95% CI = 1.02-53.95). Salmonella (S.) Bovismorbificans isolated from eight stool and one tap water samples had identical PFGE profiles. No resistant isolates were identified. Residual chlorine levels were lower than the acceptable limits before and during the outbreak. We advised consumption of bottled water and adherence to strict hand hygiene rules until tap water was declared suitable for drinking. CONCLUSIONS: Epidemiological and molecular data revealed a waterborne S. Bovismorbificans outbreak in town A. We recommend local water safety authorities to ensure that residual chlorine levels comply with the legislation towards water safety planning, to mitigate risks.

Phenotypic and molecular characterization of blaCTX-M-3 and blaCTX-M-55-producing monophasic Salmonella enterica serovar Typhimurium in Greece.

OBJECTIVES: To characterize three Salmonella enterica serovar Typhimurium strains using whole genome sequencing (WGS) and conventional methods. The isolates were recovered from three pediatric patients in Greece as part of the hospital's epidemiological surveillance system during 2016 to 2018. METHODS: Bacterial identification and antimicrobial susceptibility testing was performed using the VITEK 2 automated system, disc diffusion test, and MIC gradient test while serotyping by the slide agglutination method. Detection of resistance genes, eBurst groups (eBG), assignment to sequence types, single nucleotide polymorphism (SNP) typing, location and characterization of drug resistance regions, and in silico plasmid detection were carried out using WGS. RESULTS: All strains were identified as S. Typhimurium-monophasic, ST34, eBG1 with antigenic formula 1,4, [5], 12:i:-. They were phenotypically resistant to most antibiotics tested except piperacillin/tazobactam, imipenem, and co-trimoxazole. WGS revealed the chromosomally located genes encoding the ASSuT (ampicillin, streptomycin, sulfonamides, and tetracycline) resistant pattern in all three strains. WGS revealed extended spectrum ß-lactamase (ESBL) production in all three strains, the presence of blaCTX-M-3 on an IncI1 plasmid in two strains isolated in 2018, and the chromosomally encoded blaCTX-M-55 plus qnrS1 (resistance to ciprofloxacin) in the strain isolated in 2016. The two strains from 2018 were isolated from the same hospital ward and were genetically related. CONCLUSIONS: The emergence of ESBL among S. 1,4,[5], 12:i:- is worrisome due to its increasing antimicrobial resistance phenotype, making clinical treatment difficult. WGS provides an alternative to traditional methods of identification and genomic characterisation of strains, and serves to better understand their epidemiological dynamics and bacterial pathogenesis.

COVID-19 and Antimicrobial Resistance: Data from the Greek Electronic System for the Surveillance of Antimicrobial Resistance-WHONET-Greece (January 2018-March 2021).

Changes in hospitals' daily practice due to COVID-19 pandemic may have an impact on antimicrobial resistance (AMR). We aimed to assess this possible impact as captured by the Greek Electronic System for the Surveillance of Antimicrobial Resistance (WHONET-Greece). Routine susceptibility data of 17,837 Gram-negative and Gram-positive bacterial isolates from blood and respiratory specimens of hospitalized patients in nine COVID-19 tertiary hospitals were used in order to identify potential differences in AMR trends in the last three years, divided into two periods, January 2018-March 2020 and April 2020-March 2021. Interrupted time-series analysis was used to evaluate differences in the trends of non-susceptibility before and after the changes due to COVID-19. We found significant differences in the slope of non-susceptibility trends of Acinetobacter baumannii blood and respiratory isolates to amikacin, tigecycline and colistin; of Klebsiella pneumoniae blood and respiratory isolates to meropenem and tigecycline; and of Pseudomonas aeruginosa respiratory isolates to imipenem, meropenem and levofloxacin. Additionally, we found significant differences in the slope of non-susceptibility trends of Staphylococcus aureus isolates to oxacillin and of Enterococcus faecium isolates to glycopeptides. Assessing in this early stage, through surveillance of routine laboratory data, the way a new global threat like COVID-19 could affect an already ongoing pandemic like AMR provides useful information for prompt action.

Diversity and Resistance Profiles of Human Non-typhoidal Salmonella spp. in Greece, 2003-2020.

Salmonella spp. is one of the most common foodborne pathogens in humans. Here, we summarize the laboratory surveillance data of human non-typhoidal salmonellosis in Greece for 2003-2020. The total number of samples declined over the study period (p < 0.001). Of the 193 identified serotypes, S. Enteritidis was the most common (52.8%), followed by S. Typhimurium (11.5%), monophasic S. Typhimurium 1,4,[5],12:i:- (4.4%), S. Bovismorbificans (3.4%) and S. Oranienburg (2.4%). The isolation rate of S. Enteritidis declined (p < 0.001), followed by an increase of the less common serotypes. Monophasic S. Typhimurium has been among the five most frequently identified serotypes every year since it was first identified in 2007. Overall, Salmonella isolates were resistant to penicillins (11%); aminoglycosides (15%); tetracyclines (12%); miscellaneous agents (sulphonamides, trimethoprim, chloramphenicol and streptomycin) (12%) and third-generation cephalosporins (2%). No isolate was resistant to carbapenems. In total, 2070 isolates (24%) were resistant to one or two antimicrobial classes and 903 (10%) to three and more. Out of the 1166 isolates resistant to fluoroquinolones (13%), 845 (72%) were S. Enteritidis. S. Enteritidis was also the most frequently identified serotype with a resistance to third-generation cephalosporins (37%, 62/166), followed by S. Typhimurium (12%, 20/166). MDR was most frequently identified for S. Typhimurium and its monophasic variant (resistant phenotype of ampicillin, streptomycin, tetracycline and sulphamethoxazole with or without chloramphenicol or trimethoprim).

The Role of Coronavirus RNA-Processing Enzymes in Innate Immune Evasion.

Viral RNA sensing triggers innate antiviral responses in humans by stimulating signaling pathways that include crucial antiviral genes such as interferon. RNA viruses have evolved strategies to inhibit or escape these mechanisms. Coronaviruses use multiple enzymes to synthesize, modify, and process their genomic RNA and sub-genomic RNAs. These include Nsp15 and Nsp16, whose respective roles in RNA capping and dsRNA degradation play a crucial role in coronavirus escape from immune surveillance. Evolutionary studies on coronaviruses demonstrate that genome expansion in Nidoviruses was promoted by the emergence of Nsp14-ExoN activity and led to the acquisition of Nsp15- and Nsp16-RNA-processing activities. In this review, we discuss the main RNA-sensing mechanisms in humans as well as recent structural, functional, and evolutionary insights into coronavirus Nsp15 and Nsp16 with a view to potential antiviral strategies.

Waterborne outbreak in a rural area in Greece during the COVID-19 pandemic: contribution of community pharmacies.

INTRODUCTION: On 15 June 2020, the National Public Health Organization was informed about the identification of two cases of Escherichia coli Ο157 infection in a small town in the Peloponnese Region in Greece and we suspected an outbreak. METHODS: We asked the local pharmacist to assist us to verify the outbreak by providing the daily number of over-the-counter anti-diarrheal drugs sold from 20 May 2020 onwards. The pharmacist asked customers with gastroenteritis to submit stool samples at the local hospital. Samples were tested for 22 pathogens. We conducted a 1 : 1 case-control study. Cases and controls were retrieved from the pharmacy client list. Chlorination records of the water supply system were retrieved, and water samples were tested for microbiological indicators and viruses. RESULTS: The increased number of sales of anti-diarrheal drugs verified the outbreak. Overall, 58 cases and 57 controls were recruited for the study. Tap water consumption (odds ratio (OR)=10.9, 95% confidence interval (CI)=3.1-38.0, p<0.001) and consumption of ice cubes made from tap water (OR=39.3, 95%CI=10.3-150.9, p<0.001) were independently associated with gastroenteritis occurrence. Eleven stool samples were tested; one was positive for shigatoxin-producing E. coli, one for enteropathogenic E. coli, four for E. coli Ο157 and one for Salmonella spp. Four samples tested negative. Five water samples collected on 18 June tested negative. The residual chlorine on 5 and 14 June ranged from 0.12 mg/L to 0.14 mg/L. CONCLUSION: This was the first investigation of a waterborne outbreak in Greece performed with the collaboration of a local pharmacy. The COVID-19 pandemic favored the use of alternative resources and channels of communication with the local population, which can also be used in the future, especially in remote areas of the country.

The Rising Burden of Salmonellosis Caused by Monophasic Salmonella Typhimurium (1,4,[5],12:i:-) in Greece and New Food Vehicles.

Monophasic Salmonella typhimurium is of increasing importance worldwide. Here we present the available data regarding monophasic S. typhimurium from 2007 to 2019 in Greece, in order to assess its public health impact. Surveillance data, data on antimicrobial resistance, molecular typing by pulsed-field gel electrophoresis (PFGE), and results of the investigation of monophasic S. typhimurium outbreaks were analyzed. Overall, 403 cases were identified; 329 (81.6%) sporadic and 74 (18.4%) related to two community outbreaks in 2017. A total of 305 isolates from sporadic cases tested for antimicrobial resistance revealed resistance to ampicillin, streptomycin, sulphamethoxazole, and tetracycline (41.3%). Some 23.3% were further resistant to trimethoprim and 5.2% were also resistant to chloramphenicol. Outbreak 1 in 2017 with 37 identified cases was attributed to the consumption of raw milk from a vending machine and isolates were resistant to ampicillin, streptomycin, sulphamethoxazole, tetracycline, and trimethoprim. Outbreak 2 also with 37 cases was attributed to the consumption of pork and isolates were resistant to the five above mentioned antibiotics plus chloramphenicol. The number of human monophasic S. typhimurium isolates is low; however, since 2009, it has been among the five most frequently identified serotypes in Greece. Investigation of the outbreaks revealed that other vehicles apart from pork may be implicated in the occurrence of outbreaks.

Impact of Salmonella Control Programmes in Poultry on Human Salmonellosis Burden in Greece.

Since 2008, veterinary authorities in Greece have implemented national control programmes (NSCPs) targeting S. Enteritidis (SE) and S. Typhimurium (ST) in poultry. We assessed the effect of the programs on the reported number of human isolates. Using monthly data for 2006-2017, we defined two groups (SE, ST) and one control group with serotypes unrelated to poultry or eggs. For SE we also analysed data for 2006-2015 due to a multi-county SE outbreak in 2016. We performed an interrupted time series analysis and used a negative binominal regression model. For both SE and ST, there was no significant trend of the isolation rate before or after NSCPs' introduction. After the NSCPs' introduction there was an increasing rate (IRR: 1.005, 95% CI: 1.001-1.008) for control serotypes and a decreasing one for SE (IRR: 0.990, 95% CI: 0.986-0.995) (for 2009 to 2015 analysis). From 2006 to 2017, NSCPs had a statistically significant impact on the number of SE isolates that decreased by 49% (IRR:0.511, 95% CI: 0.353-0.739). No impact was shown on the number of ST (p-value = 0.741) and control isolates (p = 0.069). As a conclusion, NSCP's implementation was associated with decreased SE isolates and overall burden of salmonellosis; however further measures aiming at human salmonellosis due to ST, should be considered.

Public health and clinical implications of multidrug-resistant shigellosis cases in a reception centre for refugees/migrants, Greece, October-December 2019.

From October to December 2019, 18 shigellosis cases (median age: 4.3 years, range: 0-21) were identified in the Reception Center for refugees/migrants on the Greek island of Samos. Fifteen cases (83.3%) were Afghani. Median time from arrival to symptoms onset was 53 days (40-101). Isolates from 14 cases, serotyped as Shigella flexneri 1b, and from three cases, serotyped as S. sonnei phase S (I), presented a multidrug-resistant phenotype. S. flexneri 1b isolates also produced extended-spectrum ß-lactamases. Shigella flexneri 2a isolate from the remaining case was resistant to sulfomethoxazole, trimethoprim and pefloxacin. Improvement of hygiene and strengthening of laboratory investigation is needed.

Neurolisteriosis in a previously asymptomatic patient with serum IgM deficiency: a case report.

BACKGROUND: Listeria monocytogenes is an opportunistic pathogen of the central nervous system commonly associated with impaired cell-mediated immunity. We hereby present a case of adult neurolisteriosis where the only immunological feature persistently present was serum IgM deficiency, suggesting that non-specific humoral immunity may also play a central role in the control of neuroinvasion by Listeria monocytogenes. CASE PRESENTATION: A 62-year-old male who had never experienced severe infections presented with headache, nuchal rigidity and confusion. Neuroimaging was normal and lumbar puncture revealed pleiocytosis (760 leukocytes/mm3) and hypoglycorrhachia (34 mg/dL). The patient was treated empirically for bacterial meningitis. Indeed, further analysis of the CSF showed infection by Listeria monocytogenes, which was accompanied by reduced serum IgM levels that persisted well beyond the period of acute bacterial infection. Levels of IgG and IgA isotypes, along with peripheral blood counts of major leukocyte subsets, were at the same time largely preserved. Intriguingly, the absence of membrane-bound IgM on B cells was essentially complete in the acute post-infection period leading to a remarkable recovery after 12 months, suggesting that mechanisms other than defective membrane expression are underlying serum deficiency. CONCLUSIONS: As far as we know, this is the first reported case of neurolisteriosis associated with IgM deficiency in an adult individual without a history of severe infections or other underlying conditions. A possible role of circulating IgM against invasive disease caused by Listeria monocytogenes, particularly in the early course of host-pathogen interaction, is discussed.

Epidemiological Data, Serovar Distribution and Antimicrobial Resistance Patterns of Salmonella Species in Children, Greece 2011-2017: A Retrospective Study.

OBJECTIVE: This study aimed to describe Salmonella epidemiology and antimicrobial resistance in Greek children over the period of 2011-2017. MATERIALS AND METHODS: A 7-year retrospective study (2011-2017) was performed, based on data recorded by the National Reference Centre for Salmonella, among children aged ≤14 years. Epidemiological data, serovar distribution and antimicrobial resistance patterns were recorded. RESULTS: Overall, 2347 Salmonella isolates were collected (27 typhoid-paratyphoid). Salmonellosis cases increased by almost 2-fold in 2017 compared to 2011. The highest rates were reported in August, with infants being the most vulnerable group (17.9%). The majority of isolates were identified in stool samples (91%). Boys slightly outnumbered girls (˜1.05:1). Salmonella Enteritidis was the most prevalent serovar (28.5%), followed by Salmonella Typhimurium (12.2%) and Salmonella monophasic Typhimurium (10.4%). Non-typhoid isolates displayed low resistance rates to 3rd generation cephalosporins (1%) and ciprofloxacin (0.3%), while the corresponding resistance of typhoid isolates was 10% and 5% respectively. An increasing trend of Salmonella monophasic Typhimurium was recorded, associated with high rates of multidrug resistance, reaching a percentage of 97.8% in 2017. CONCLUSIONS: Salmonellosis epidemiology in Greek children is comparable to previously published European data. Antimicrobial resistance rates to 3rd-generation cephalosporins and ciprofloxacin for non-typhoid and typhoid-paratyphoid remain low. Notably, there is an increasing prevalence of Salmonella monophasic Typhimurium isolates, associated with multiple antimicrobial resistance.

Salmonellosis outbreak with novel Salmonella enterica subspecies enterica serotype (11:z41:e,n,z15) attributable to sesame products in five European countries, 2016 to 2017.

In spring 2016, Greece reported an outbreak caused by a previously undescribed Salmonella enterica subsp. enterica serotype (antigenic formula 11:z41:e,n,z15) via the Epidemic Intelligence Information System for Food- and Waterborne Diseases and Zoonoses (EPIS-FWD), with epidemiological evidence for sesame products as presumptive vehicle. Subsequently, Germany, Czech Republic, Luxembourg and the United Kingdom (UK) reported infections with this novel serotype via EPIS-FWD. Concerned countries in collaboration with the European Centre for Disease Prevention and Control (ECDC) and European Food Safety Authority (EFSA) adopted a common outbreak case definition. An outbreak case was defined as a laboratory-confirmed notification of the novel Salmonella serotype. Between March 2016 and April 2017, 47 outbreak cases were notified (Greece: n = 22; Germany: n = 13; Czech Republic: n = 5; Luxembourg: n = 4; UK: n = 3). Whole genome sequencing revealed the very close genetic relatedness of isolates from all affected countries. Interviews focusing on sesame product consumption, suspicious food item testing and trace-back analysis following Salmonella spp. detection in food products identified a company in Greece where sesame seeds from different countries were processed. Through European collaboration, it was possible to identify and recall sesame spread as one contaminated food item serving as vehicle of infection and trace it back to its origin.

Multiple clones and low antimicrobial resistance rates for Salmonella enterica serovar Infantis populations in Greece.

All the Salmonella enterica ser. Infantis strains isolated under official control programs in Greece during a four year period were studied, 23 of human origin, 16 from food animals and one from food. Molecular analyses (PFGE) in combination with antimicrobial susceptibility testing were used to study whether the occurrence S. Infantis in Greece resulted from different biotypes or a successful spread of one clone. Low rates of antimicrobial resistance were observed, except for streptomycin among human isolates (48%), indicating that selective pressure due to consumption of antimicrobials has not resulted the spread of dominant clones. Pulsed Field Gel Electrophoresis revealed 31 XbaI distinct pulsotypes among the 40 strains with 60% overall similarity reflecting diversity. Four main clusters were constructed, using an 85% cut off value, clusters A, B, C and D consisting of 14, 6, 8 and 8 isolates respectively. Point source of transmission was not hypothesized as multiple reservoirs of the serovar seem to be present in Greece during the study period.

An outbreak of a possibly new Salmonella enterica subspecies enterica serovar with the antigenic formula 11:z41:e,n,z15, Greece, March to May 2016: preliminary results.

Eleven Salmonella spp. isolates with the antigenic type 11:z41:e,n,z15 - not referred to in the 9th edition of the White-Kauffman-Le Minor Scheme - were identified at the National Reference Laboratory for Salmonella in Greece. Their pulsed-field gel electrophoresis profiles were indistinguishable. No apparent epidemiological link has yet been identified; the results of a case-case study are pending.

Evaluation of underreporting of salmonellosis and shigellosis hospitalised cases in Greece, 2011: results of a capture-recapture study and a hospital registry review.

BACKGROUND: Salmonellosis and shigellosis are mandatorily notifiable diseases in Greece. Underreporting of both diseases has been postulated but there has not been any national study to quantify it. The objective of this study was to: a) estimate underreporting of hospitalised cases at public Greek hospitals in 2011 with a capture-recapture (C-RC) study, b) evaluate the accuracy of this estimation, c) investigate the possible impact of specific factors on notification rates, and d) estimate community incidence of both diseases. METHODS: The mandatory notification system database and the database of the National Reference Laboratory for Salmonella and Shigella (NRLSS) were used in the C-RC study. The estimated total number of cases was compared with the actual number found by using the hospital records of the microbiological laboratories. Underreporting was also estimated by patients' age-group, sex, type of hospital, region and month of notification. Assessment of the community incidence was based on the extrapolation of the hospitalisation rate of the diseases in Europe. RESULTS: The estimated underreporting of salmonellosis and shigellosis cases through the C-RC study was 47.7% and 52.0%, respectively. The reporting rate of salmonellosis significantly varied between the thirteen regions of the country from 8.3% to 95.6% (median: 28.4%). Age and sex were not related to the probability of reporting. The notification rate did not significantly differ between urban and rural areas, however, large university hospitals had a higher underreporting rate than district hospitals (p-value < 0.001). The actual underreporting, based on the hospital records review, was close to the estimated via the C-RC study; 52.8% for salmonellosis and 58.4% for shigellosis. The predicted community incidence of salmonellosis ranged from 312 to 936 and of shigellosis from 35 to 104 cases per 100,000 population. CONCLUSIONS: Underreporting was higher than that reported by other countries and factors associated with underreporting should be further explored. C-RC analysis seems to be a useful tool for the assessment of the underreporting of hospitalised cases. National data on underreporting and under-ascertainment rate are needed for assessing the accuracy of the estimation of the community burden of the diseases.

Prevalence study of Legionella spp. contamination in Greek hospitals.

Water and swab samples were collected from 13 hospitals and analyzed for Legionella counts. Legionella was detected in eight out of 13 hospitals and in 22 of 130 water and swab-collected samples. A total of 72.7% of the strains were L. pneumophila ser. 1, 22.7% were L. pneumophila ser. 2-14, and 4.5% did not belong to any of these groups. AFLP typing of the L. pneumophila ser. 1 strains generated two distinguishable AFLP types. There was no significant correlation to the sample type with Legionella recovery. Legionella isolation was more likely to occur in the cooling towers than the water system. Water temperatures of 30-40 degrees C seem to favor Legionella growth. Of the 265 serum samples taken from the medical and technical staff for the control of IgG titre, 89.4% were negative, 7.2% were positive, and for 3.4% the result was doubtful. No association between IgG titre and maximum observed level of Legionella occurrence was detected.

Correlation between bacterial indicators and bacteriophages in sewage and sludge.

The use of bacteriophages as potential indicators of faecal pollution has recently been studied. The correlation of the number of bacterial indicators and the presence of three groups of bacteriophages, namely somatic coliphages (SOMCPH), F-RNA-specific phages (FRNAPH) and phages of Bacteroides fragilis (BFRPH), in raw and treated wastewater and sludge is presented in this study. Raw and treated wastewater and sewage sludge samples from two wastewater treatment plants in Athens were collected on a monthly basis, over a 2-year period, and analysed for total coliforms, Escherichia coli, intestinal enterococci and the three groups of bacteriophages. A clear correlation between the number of bacterial indicators and the presence of bacteriophages was observed. SOMCPH may be used as additional indicators, because of their high densities and resistance to various treatment steps.

PCR detection of Salmonella spp. using primers targeting the quorum sensing gene sdiA.

Bacteria communicate with one another and with their host using chemical signalling molecules. This phenomenon is generally described as quorum sensing. A set of primers for PCR detection of Salmonella spp. has been designed using as target the sdiA gene which encodes a signal receptor of the LuxR family. The PCR product (274 bp) was confirmed by sequencing. A number of 81 non-Salmonella strains (representing 24 different species) were tested and gave negative results, while a total of 101 different serotypes of Salmonella (155 strains) tested positive for the presence of the sdiA gene. The sensitivity and specificity of the sdiA-based PCR assay were also checked in artificially contaminated human faecal samples. In this study, we demonstrate that quorum sensing genes can be successfully exploited as diagnostic markers.