Zoonotic Foodborne Infections in a Tertiary Healthcare Setting: Clinical and Epidemiological Aspects of Campylobacter and Salmonella Infection in Northern Portugal in 2010–2020

Abstract

This study aimed to describe the epidemiology, clinical presentation, and management of Campylobacter sp. and Salmonella sp. infections in a tertiary hospital in Northern Portugal (2010–2020) and identify factors associated with reporting, invasive disease, and hospitalization. A retrospective review included patients with Campylobacter sp. (2015–2020) or Salmonella sp. infections (2010–2020). Reported cases were cross-referenced with the National Epidemiological Surveillance System. A total of 742 patients were included: 342 with Campylobacter sp., 392 with non-typhoidal Salmonella (NTS), and 15 with typhoidal Salmonella (TS). Immunosuppression was present in 16.7% of cases. NTS infections were invasive in 21.4% of cases, compared to 4.0% for Campylobacter. Macrolide resistance in Campylobacter reached 10%, particularly in C. coli, while multidrug resistance (MDR) was observed in 37.8% of NTS isolates. Immunocompromised patients accounted for over 70% of relapses in Campylobacter disease. Reporting rates were low: 32.8% for NTS, 30.6% for Campylobacter, and 14.3% for TS. Reporting was associated with male sex, no immunosuppression, hospitalization, and non-MDR organisms. In conclusion, immunocompromised patients face higher risks of invasive and relapsing disease. High rates of MDR in NTS limit treatment options. Underreporting remains significant, underscoring the need for improved awareness and reporting to inform public health strategies.

1. Introduction

Campylobacter sp. and Salmonella sp. are zoonotic foodborne agents and two of the most common infectious causes of gastroenteritis worldwide [1,2]. These infections represent a significant impact on global morbidity and economic burden. Infection by these agents is usually benign, presenting as acute gastroenteritis [2], but both have been implicated in gastrointestinal acute complications (such as toxic megacolon, perforation, and hemorrhage—[3,4,5,6,7]) and invasive disease, with bacteremia and extraintestinal metastatic infections, including meningitis, bone and joint infections, endovascular infections, and endocarditis [8]. In this setting, mortality can be as high as 15% [9]. Additionally, infection by Campylobacter sp. has been associated with autoimmune and inflammatory conditions such as Guillain–Barré syndrome (GBS), inflammatory intestinal disease, irritable bowel syndrome and celiac disease [1]. Reactive arthritis is present in 3–13% of C. jejuni-infected patients [10] and in 0.07% of GBS cases [11], making this species the most commonly recognized infectious cause of the syndrome, being responsible for 5–41% of cases [12]. Invasive infections by non-typhoidal Salmonella sp. (NTS) and Campylobacter sp. in the European setting have mostly been linked to old age and immunosuppression [13,14]. For NTS, some of the most extensively studied types of immunosuppression include solid and hematologic malignancies [15] and chronic immunosuppressive therapy, especially anti-TNF-α drugs [16]. HIV infection/AIDS has been linked with recurrent invasive NTS infection [17]. Hypogammaglobulinemia, either primary or secondary, seems to be a risk factor for invasive and recurrent campylobacteriosis [18].

In the European region, campylobacteriosis and salmonellosis are the two most commonly reported gastrointestinal infections and important causes of foodborne disease outbreaks. Their hospitalization rate is significant (21.0–29.9%), but mortality is considerably low (0.05–0.19%). At a global level, the more frequently identified Campylobacter species are C. jejuni (88.1%), C. coli (10.6%), C. fetus (0.16%), C. lari (0.11%), and C. upsaliensis (0.09%) [19]. Three Salmonella enterica subsp. enterica serovars accounted for 70.3% of human cases in 2019: S. enteritidis (48.7%), S. typhimurium (12.4%), and monophasic S. typhimurium 1,4,[5],12:i:- (11.1%) [19]. Resistance to quinolones is common in Europe both in Campylobacter (60–70%) and Salmonella (around 15%) [20].

However, marked differences are reported between European countries. In Portugal, systematic declaration of cases of salmonellosis was implemented before 2010, but declaration of campylobacteriosis cases to the National Epidemiological Surveillance System (SINAVE, from the Portuguese Sistema Nacional de Vigilância Epidemiológica) only started in 2015. Estimates of the incidence of Campylobacter sp. infection and clinical descriptions of cases before this period are scarce and dispersed, with only a few studies being published during that time. These studies [21,22,23,24,25,26] showed positivity rates from diarrhea samples of 5.3–31.9% for Campylobacter sp. and 14.8–19.4% for Salmonella sp., both predominantly isolated in children under 5 years old. Rates of isolation showed an increasing trend for Campylobacter sp. but a stability for Salmonella sp. C. jejuni was the most commonly identified species, followed by C. coli or C. concisus, depending on the studies. Resistance to quinolones in Campylobacter sp. was generally >50% (and >90% in the most recent studies) and resistance to macrolides was 6.5–20%; rising trends of resistance for both antibiotic classes were noted. Most of these studies comprised a small temporal scale and identified fewer than 150 patients with Campylobacter sp. or non-typhoidal Salmonella sp. infection. Studies of a larger magnitude have been conducted in other countries and are needed in Portugal [27].

In Portugal, systematic surveillance after 2015 and additional hospital-based studies have been suggesting similar findings: macrolide resistance in Campylobacter sp. exceeds 10% [28]; combined resistance to macrolides and fluoroquinoles in human isolates is one of the highest in European countries, especially in C. coli (36.8%) [20]; and incidence rates show an increasing trend for Campylobacter sp. and stable for Salmonella sp. [19]. Even so, incidence rates estimated by the number of cases officially reported were much lower than the average in the EU (7.7 vs. 40.3 confirmed cases/100,000 population for Campylobacter sp. and 2.5 vs. 13.7 for Salmonella sp.) and the incidences calculated for neighboring countries [19]. On the other hand, Portugal reported one of the highest hospitalization rates in 2017 (84%). These differences likely reflect distinct reporting practices, leading to a greater underreporting of mild disease and a primary focus on severe disease. This hypothesis has been supported by studies on gastroenteritis in returned travelers [29] and modeling studies [30] that suggest only 1/2080 to 1/93 cases of salmonellosis are reported to the national monitoring system, putting Portugal among the European countries with the highest true incidences and simultaneously making it one of the countries with highest underreporting rates.

The objectives of this study are to describe the epidemiology, clinical presentation, and management of Campylobacter sp. and Salmonella sp. infection in a tertiary hospital in Northern Portugal between 2010 and 2020 and to determine reporting frequency and identify factors associated with underreporting of cases. This study also aims to investigate risk factors for invasive disease and hospitalization, and to compare these outcomes according to Campylobacter sp./Salmonella sp. species/serovar.

2. Materials and Methods

2.1. Study Population and Elegibility Criteria

A single-center retrospective study was conducted in São João University Hospital, a tertiary public healthcare provider in the Oporto metropolitan area, in Northern Portugal (a country located in Southwest Europe, bordering Spain and the Atlantic Ocean). Patients diagnosed with Campylobacter sp. or Salmonella sp. infections were included in the study. The period of analysis for this study was 2010–2020 for Salmonella sp. and 2015–2020 for Campylobacter sp., inclusively.

2.2. Microbiological Diagnosis

Patients were retrospectively identified by the Clinical Pathology department via a database search. Positive microbiological culture results were screened automatically for the keywords “Salmonella” and “Campylobacter”. Regarding the cultural exam protocol, since 2016, stool samples have been systematically sent to the laboratory in a transport medium (ETM^® Enteric Transport System—Alpha-Tec). Selective culture media were used for Campylobacter sp. and Salmonella sp. Since 2019, incubation at 42 °C with a microaerophilic atmosphere has been implemented for Campylobacter sp. isolation. Identification of Salmonella sp. was performed using VITEK^® 2 GN (bioMérieux Inc., Durham, NC, United States of America [USA]) identification cards and identification of Campylobacter sp. was performed by matrix-assisted laser desorption ionization–time-of-flight mass spectrometry (using VITEK^® MS). Antimicrobial susceptibility testing was performed using the automated VITEK^® 2 System for Salmonella sp. and by disk diffusion for Campylobacter sp., with interpretation according to the most updated clinical breakpoints of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) at the time. Serotyping of Salmonella enterica isolates was performed by sera agglutination tests using BIORAD^® (Hercules, CA, USA) kits. A list of serological testing performed in the Hospital was similarly obtained and the positive results were selected for analysis. The search for antibodies against Salmonella Typhi or Paratyphi was performed using the Widal indirect agglutination test and a positive result was admitted when antibody titers against O or H antigen increased by at least four-fold between serum samples taken two or more weeks apart. Finally, a list of samples where Campylobacter sp. DNA was identified by real-time polymerase chain reaction (PCR) was retrieved. Commercial Campylobacter PCR Detection kits (Bio-Rad^®) were used. All types of samples were included in this search, regardless of the department where they were collected. Patients with one or more positive results (culture, serology, and/or PCR) were considered infected by Salmonella sp. and/or Campylobacter sp. and were included in the study.

2.3. Data Collection, Variable Definitions, and Grouping

Data from the patients were extracted directly from the medical records respective to the episodes when Campylobacter sp./Salmonella sp. infection was diagnosed through a standardized procedure. Sociodemographic and clinical information was retrieved for each patient, including clinical presentation (signs/symptoms), underlying conditions, diagnosis, management, and outcome. Data of the strains isolated by the microbiology laboratory were also collected, (sub)species were identified (and serovar for Salmonella enterica subsp. enterica), and antimicrobial susceptibility testing was conducted. Simultaneously, a list of the cases reported to the National Epidemiological Surveillance System by professionals of the São João University Hospital was compiled.

Categorical variables extracted from the questionnaire were analyzed mostly using the original categories provided as answer options, but regrouping was performed in some cases. Recent travel was defined as traveling outside Portugal in the 12 months prior to the diagnosis. Fever was defined as body temperature above 38 °C (or assumed when written in the records). Acute kidney injury was defined according to the 2012 KDIGO Clinical Practice Guideline [31]. Bacteremia was defined as Campylobacter sp./Salmonella sp. isolation from ≥1 blood culture. Extraintestinal focal infection was defined as isolation from ≥1 samples other than feces (or other intestinal material) and blood, plus a compatible clinical picture. Antibiotic treatment was considered appropriate if the infecting strain was susceptible to ≥1 of the drugs administered. Empirical therapy was based on clinical data only, without any microbiological results. Targeted therapy was based on the results of culture and susceptibility testing. Failure was defined as persistence of symptoms and positive culture/PCR, despite appropriate therapy. Relapse was defined by recurrence of signs/symptoms and/or positive culture/PCR after initial clinical improvement and negative diagnostic tests. Outcome was assessed at the moment of discharge or death.

2.4. Statistical Analysis

Statistical analysis, including absolute and relative frequencies and hypothesis testing, was performed using IBM^® SPSS^® Statistics Version 29.0. Descriptive statistics are expressed as absolute frequencies and percentages for categorical variables and as means with standard deviations or medians with interquartile ranges (IQRs) for continuous variables. Comparisons between groups were performed using Pearson’s χ² test for categorical variables (or Fisher’s exact in case of failure of the assumptions of the χ² test). For continuous variables, after checking the assumptions of normality and homogeneity of the variances, instead of t-test and ANOVA, the Mann–Whitney U test was used for comparing two independent groups. A value of p < 0.05 was considered statistically significant. Multivariate analyses were conducted to identify factors associated with invasive disease, hospitalization, and underreporting. These analyses were performed through multiple binary logistic regression models, analyzing variables with statistical meaning in the univariate analysis (p < 0.20) and some biologically relevant or potentially confounding variables. For those variables that remained significant, crude odds ratios (ORs) were updated to adjusted odds ratios (aORs) with a 95% confidence interval (CI). The reference categories used for each independent variable are specified in each multivariate analysis results table.

3. Results

3.1. Sociodemographic Data

In total, 742 patients were included in this study: 342 infected with Campylobacter sp., 392 with NTS, and 15 with typhoidal Salmonella (TS). Campylobacter jejuni and Salmonella typhimurium were the most common agents. Cases of coinfection were detected: C. jejuni + C. coli (n = 4); C. jejuni + S. typhimurium (n = 2); and C. jejuni + S. paratyphi C (n = 1). The distribution of cases by species/serovar in total and by year of diagnosis is represented in Figure 1 and the percentage of cases diagnosed by municipality between 2010 and 2020 is represented in Figure 2. Additionally, Figure 3a presents the number of cases diagnosed by month, showing a clear peak in cases of NTS between August and October, but no clear seasonality for Campylobacter infections.

Median age was significantly different between Campylobacter and NTS cases (4 vs. 9 years old, p < 0.001) and within each group (older for C. coli and for S. enteritidis). A second (smaller) peak of NTS infections was detected in the elderly. Distribution of cases by age is represented in Figure 3b.

Male sex was predominant for all agents (except TS), although the percentage was significantly lower for NTS compared to Campylobacter (p = 0.008). The proportion of patients who had traveled abroad before the onset of symptoms was similar for Campylobacter and NTS (p = 0.588). Sociodemographic characteristics of patients are summarized in

Table 1. Sociodemographic aspects of cases of Campylobacter (2015–2020) and Salmonella (2010–2020) infections diagnosed in a tertiary hospital in Northern Portugal, categorized by group (Campylobacter, non-typhoidal Salmonella, and typhoidal Salmonella), and species/serovar within each group.

	Campylobacter				NTS				p Value 3	TS
	All Species	C. jejuni	C. coli	p Value 1	All Serovars	S. typhimurium	S. enteritidis	p Value 2		All Serovars
All patients (n)	342	295	31		392	246	80			15
Age, median (IQR), years	4 (1–22.25)	2 (1–15)	20 (3–49)	0.005	9 (3–59)	6 (2–44.5)	11.5 (4–62)	0.003	<0.001	27 (3–59)
Male sex (%)	62.3 (213/342)	63.4 (187/295)	61.3 (19/31)	0.818	52.6 (206/392)	52.4 (129/246)	50.0 (40/80)	0.704	0.008	40.0 (6/15)
Travel abroad (%)	1.5 (5/329)	0.7 (2/286)	6.7 (2/30)	0.005	1.1 (4/377)	1.3 (3/238)	0 (0/76)	0.599	0.588	21.4 (3/14)

Abbreviations: IQR (interquartile range), NTS (non-typhoidal Salmonella), TS (typhoidal Salmonella). ¹ Comparing Campylobacter jejuni and C. coli. ² Comparing Salmonella Typhimurium and S. enteritidis. ³ Comparing Campylobacter sp. and non-typhoidal Salmonella sp.

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3.2. Clinical Presentation

Globally, 16.7% of patients were immunocompromised, although this percentage was significantly higher for Campylobacter than for NTS infections (p = 0.003). Of the Campylobacter infections, 27.8% occurred in patients with primary immunosuppression but none of the NTS infections were diagnosed in the setting of primary immunosuppression. The type of secondary immunosuppression factor was similarly distributed in Campylobacter and NTS infections (p = 0.951); chronic immunosuppressive therapy was the most common, followed by solid malignancy. Of the infections diagnosed in the study period, 2.5% were asymptomatic (similar proportion across both agents). Among symptomatic patients, diarrhea was the most common symptom, present in over 80% of cases for every agent, with some differences: less common in NTS than in Campylobacter; less common in S. enteritidis infections than in S. typhimurium ones; and less common in C. coli than in C. jejuni. Fever and nausea/vomiting were reported in 68.2% and 39.5% of patients, respectively, and they were both more commonly seen in NTS infections than in Campylobacter infections. Rash was rarely reported (2.5% of cases). NTS infections were invasive in 21.4% of cases, of which almost half represented extraintestinal focal disease. Although bacteremia was similarly common for S. typhimurium and S. enteritidis, extraintestinal focal infection was significantly more common for S. enteritidis. For TS, extraintestinal focal infection was diagnosed in 8/12 cases. Anatomical locations and number of cases of invasive extraintestinal focal disease for Salmonella sp. infections are represented in Figure 4.

In contrast, invasive Campylobacter infection was identified in only 4.0% of cases. Acute kidney injury (AKI) was a common complication due to infection, especially for NTS (20.2%). NTS infections were more frequently hospitalized than Campylobacter infections (54.3 vs. 24.3%), although differences in hospitalization rates were noted between C. jejuni and C. coli (45.2 vs. 22.0%). The median duration of hospitalization was similar between NTS and Campylobacter cases (6 vs. 7 days, p = 0.090). Immunosuppression and clinical presentation aspects of patients are summarized in

Table 2. Immunosuppression and clinical presentation aspects of cases of Campylobacter (2015–2020) and Salmonella (2010–2020) infections diagnosed in a tertiary hospital in Northern Portugal, categorized by group (Campylobacter, non-typhoidal Salmonella, and typhoidal Salmonella), and species/serovar within each group.

	Campylobacter				NTS				p Value 3	TS
	All Species	C. jejuni	C. coli	p Value 1	All Serovars	S. typhimurium	S. enteritidis	p Value 2		All Serovars
All patients (n)	342	295	31		392	246	80			15
Immunosuppression (%)	21.1 (72/342)	17.3 (49/284)	53.3 (16/30)	<0.001	12.8 (50/392)	10.4 (25/240)	13.0 (10/77)	0.531	0.003	20.0 (3/15)
Primary (%)	27.8 (20/72) a	16.3 (8/49)	56.2 (9/16)	0.002	0 (0/50)	0 (0/25)	0 (0/10)	NA	<0.001	33.3 (1/3)
Secondary (%)	72.2 (52/72)	83.7 (41/49)	43.8 (7/16)		100 (50/50)	100 (25/25)	100 (10/10)			66.7 (2/3)
Solid malignancy	25.5 (13/51)	25.0 (10/40)	28.6 (2/7)	0.977	24.0 (12/50)	28.0 (7/25)	10.0 (1/10)	0.663	0.951	0 (0/2)
Hematologic malignancy	5.9 (3/51)	7.5 (3/40)	0 (0/7)		4.0 (2/50)	8.0 (2/25)	0 (0/10)			0 (0/2)
Solid organ transplant	19.6 (10/51)	20.0 (8/40)	28.6 (2/7)		18.0 (9/50)	16.0 (4/25)	20.0 (2/10)			50.0 (1/2)
HIV infection/AIDS	15.7 (8/51)	17.5 (7/40)	14.3 (1/7)		22.0 (11/50)	20.0 (5/25)	40.0 (4/10)			0 (0/2)
Immunosupressive therapy	33.3 (17/51) b	30.0 (12/40)	28.6 (2/7)		32.0 (16/50) c	28.0 (7/25)	30.0 (3/10)			50.0 (1/2)
Not reported	1.9 (1/52)	2.4 (1/41)	0 (0/7)		0 (0/50)	0 (0/25)	0 (0/10)			0 (0/2)
Asymptomatic (%)	3.3 (11/329)	2.4 (7/286)	3.3 (1/30)	0.769	1.8 (7/379)	2.1 (5/241)	2.6 (2/76)	0.773	0.207	0 (0/12)
Symptoms (1st episode)
Abdominal pain (%)	45.6 (145/318)	47.0 (131/279)	34.5 (10/29)	0.188	52.4 (195/372)	49.2 (116/236)	55.3 (42/76)	0.267	0.081	66.7 (8/12)
Diarrhea (%)	95.6 (304/318)	97.8 (273/279)	89.7 (26/29)	0.006	90.3 (336/372)	94.5 (223/236)	80.3 (61/76)	0.001	0.006	75.0 (9/12)
Fever (%)	63.2 (201/318)	65.9 (184/279)	44.8 (13/29)	0.022	72.0 (268/372)	76.7 (181/236)	65.8 (50/76)	0.121	0.013	83.3 (10/12)
Nausea/vomiting (%)	28.0 (89/318)	29.4 (82/279)	17.2 (5/29)	0.157	49.2 (183/372)	50.0 (118/236)	48.7 (37/76)	0.975	<0.001	41.7 (5/12)
Rash (%)	2.5 (8/318)	2.9 (8/279)	0 (0/29)	0.354	2.2 (8/372)	2.1 (5/236)	2.6 (2/76)	0.674	0.745	0 (0/12)
Invasive infection (%)	4.0 (13/328)	1.7 (5/291)	13.3 (4/30)	<0.001	21.4 (81/379)	16.7 (41/246)	23.8 (19/80)	0.156	<0.001	66.7 (8/12)
Bacteremia/sepsis (%)	3.7 (12/328)	1.4 (4/291)	13.3 (4/30)	<0.001	12.4 (47/379)	11.4 (28/246)	11.2 (9/80)	0.974	<0.001	50.0 (6/12)
Extraintestinal focal infection (%)	1.2 (4/328)	0.7 (2/291)	3.3 (1/30)	0.152	10.3 (39/379)	6.5 (16/246)	13.8 (11/80)	0.041	<0.001	16.7 (2/12)
Acute kidney injury (%)	8.1 (21/259)	7.5 (17/227)	13.6 (3/22)	0.311	20.2 (70/346)	19.2 (42/219)	20.3 (14/69)	0.839	<0.001	33.3 (4/12)
Hospitalisation (%)	24.3 (83/342)	22.0 (65/295)	45.2 (14/31)	0.004	54.3 (213/392)	54.9 (135/246)	48.8 (39/80)	0.340	<0.001	53.3 (8/15)
Duration, median (IQR), days	7 (4–14)	6 (3.5–12)	12 (8.5–15)	0.015	6 (3–10)	5 (3–10)	5 (3–9)	0.970	0.090	9 (3.25–13.25)

Abbreviations: AIDS (acquired immunodeficiency syndrome), HIV (human immunodeficiency virus), IQR (interquartile range), NA (not applicable), NTS (non-typhoidal Salmonella), TS (typhoidal Salmonella). ¹ Comparing Campylobacter jejuni and C. coli. ² Comparing Salmonella Typhimurium and S. enteritidis. ³ Comparing Campylobacter sp. and non-typhoidal Salmonella sp. ^a—10 common variable immunodeficiency, 5 X-linked agammaglobulinemia, 2 hypogammaglobulinemia, 1 Barth’s syndrome, 2 non-specified. ^b—6 corticosteroids alone, 3 corticosteroids + 2nd drug (rituximab, vedolizumab, micophenolate), 3 azathioprine, 2 infliximab, 1 methotrexate, 1 natalizumab. ^c—8 corticosteroids alone, 4 corticosteroids + 2nd drug (infliximab, methotrexate, rituximab, azathioprine), 2 azathioprine, 1 adalimumab.

.

The clinical details of the species/serovars identified during this period that are not presented in Table 2 are described below. C. fetus infections were diagnosed in seven patients. All were over 60 years old and two were immunosuppressed. Four patients presented with invasive infection with bacteremia, but none had extraintestinal focal disease. Three patients were hospitalized; none died. None of the C. fetus strains were tested for antimicrobial susceptibility. C. ureolyticus was identified in a single case, as an extraintestinal focal infection (skin abscess). During this period, a single case of S. Montevideo disease was diagnosed in a renal transplant recipient, presenting as an invasive infection (urinary tract). The remaining isolates were not identified at the species/serovar level.

3.3. Microbiological Diagnosis and Antimicrobial Resistance

Most samples where NTS or Campylobacter were identified were collected in the emergency department, in a similar proportion for the two groups (65.3 vs. 66.1%). Samples collected in the wards represented a larger share for NTS infections (21.7 vs. 12.0%), whereas collecting in the consultation was more common for Campylobacter (15.2 vs. 7.9%).

All cases of NTS infection were diagnosed using a cultural exam. Only one out of fifteen cases of TS infection was diagnosed using the Widal test (all the others were diagnosed via cultural exam). Nucleic acid amplification was used for diagnosis in 4.4% of Campylobacter infections. Among cases where identification of pathogens was performed by cultural exam, antimicrobial susceptibility testing was performed in 98.0% of NTS isolates and 48.0% of Campylobacter isolates. Resistance to fluoroquinolones was high for both Campylobacter (96.2% of isolates tested), NTS (69.3%), and TS (78.6%). Macrolide resistance in Campylobacter approached 10% and was significantly higher for C. coli (62.5%). Resistance to third-generation cephalosporins was detected in 2.3% of NTS isolates. The multidrug resistance (MDR) phenotype was present in 37.8% of NTS isolates and was significantly more common in S. typhimurium (p < 0.001). No Salmonella sp. isolates were resistant to carbapenems, but 2/125 were resistant to colistin. Microbiological diagnosis aspects of cases and antimicrobial resistance are summarized in

Table 3. Microbiological diagnosis aspects and antimicrobial resistance of cases of Campylobacter (2015–2020) and Salmonella (2010–2020) infections diagnosed in a tertiary hospital in Northern Portugal, categorized by group (Campylobacter, non-typhoidal Salmonella, and typhoidal Salmonella), and species/serovar within each group.

	Campylobacter				NTS				p Value 3	TS
	All Species	C. jejuni	C. coli	p Value 1	All Serovars	S. typhimurium	S. enteritidis	p Value 2		All Serovars
All patients (n)	342	295	31		392	246	80			15
Antimicrobial susceptibility testing
Performed (%)	45.9 (157/342)	47.5 (140/295)	51.6 (16/31)	0.660	98.0 (384/392)	99.1 (244/246)	98.8 (79/80)	0.722	<0.001	93.3 (14/15)
Resistance detected
Fluoroquinolones (%)	96.2 (151/157)	95.7 (134/140)	100 (16/16)	0.505	69.3 (253/365)	66.2 (155/234)	75.3 (55/73)	0.153	<0.001	78.6 (11/14)
Tetracyclines (%)	73.2 (115/157)	72.6 (102/140)	75.0 (12/16)	0.571
Macrolides (%)	9.7 (15/155)	3.6 (5/139)	62.5 (10/16)	<0.001
Cotrimoxazol (%)					9.9 (38/383)	8.6 (21/241)	5.1 (4/79)	0.346		21.4 (3/14)
Ampicillin (%)					63.6 (243/382)	80.7 (196/243)	22.8 (18/79)	<0.001		35.7 (5/14)
3rd generation cephalosporins (%)					2.3 (9/384)	1.6 (4/244)	3.5 (2/79)	0.610		0 (0/14)
MDR (%)	6.5 (10/155)	2.9 (4/139)	37.5 (6/16)	<0.001	37.8 (145/384)	46.7 (114/244)	12.7 (10/79)	<0.001	<0.001	28.6 (4/14)

Abbreviations: MDR (multidrug resistance), NTS (non-typhoidal Salmonella), TS (typhoidal Salmonella). ¹ Comparing Campylobacter jejuni and C. coli. ² Comparing Salmonella Typhimurium and S. enteritidis. ³ Comparing Campylobacter sp. and non-typhoidal Salmonella sp.

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3.4. Management and Outcome

Approximately 40% of patients received antibiotic treatment for their Campylobacter or NTS infection. In total, 90% of patients with TS infection were treated. Monotherapy regimens were used in around 90% of antibiotic treated patients, regardless of species/serovar. Macrolides were the most used class to treat Campylobacter infections, followed by fluoroquinolones (in 17.4% of cases). Third-generation cephalosporins were the preferred agents for NTS infections, followed by fluoroquinolones (28.5%). The antibiotic regimen was switched in 8.9% of Campylobacter cases and 14.7% of NTS cases, most commonly after genus/species/serovar identification. Failure of treatment was described in 2.4% of Campylobacter and 2.5% of NTS cases (p = 0.986) and death occurred in 2.6% of Campylobacter and 3.5% of NTS cases (p = 0.597). Symptomatic relapses were identified in 4.2% of Campylobacter-infected patients and 1.5% of NTS (p = 0.030) and were often single. Times from first or previous episode to relapse ranged from 0.5 to 23 months and were significantly longer for Campylobacter infections (median 7.0 vs. 1.8 months, p = 0.014). Over 70% of relapses of Campylobacter disease were diagnosed in immunocompromised patients. Overall, 77.8% of relapses were treated, and a different antibiotic drug was used in 76.2% of these cases. The management and outcome aspects of cases are summarized in

Table 4. Management and outcome aspects of cases of Campylobacter (2015–2020) and Salmonella (2010–2020) infections diagnosed in a tertiary hospital in Northern Portugal, categorized by group (Campylobacter, non-typhoidal Salmonella, and typhoidal Salmonella), and species/serovar within each group.

	Campylobacter				NTS				p Value 3	TS
	All Species	C. jejuni	C. coli	p Value 1	All Serovars	S. typhimurium	S. enteritidis	p Value 2		All Serovars
All patients (n)	342	295	31		392	246	80			15
Antibiotic treatment (%)	38.5 (124/322)	34.4 (96/279)	63.3 (19/30)	0.003	43.6 (163/374)	38.2 (91/238)	43.2 (32/74)	0.496	0.190	90.0 (9/10)
Monotherapy	87.9 (109/124)	88.5 (85/96)	84.2 (16/19)	0.510	88.3 (144/163)	87.9 (80/91)	84.4 (27/32)	0.609	0.943	88.8 (8/9)
Macrolide	47.7 (52/109)	52.9 (45/85)	43.8 (7/16)	0.500	4.5 (7/144)	5.0 (4/80)	7.4 (2/27)	0.641	<0.001	0 (0/8)
Fluoroquinolone	17.4 (19/109)	16.5 (14/85)	6.2 (1/16)	0.292	28.5 (41/144)	22.5 (18/80)	37.0 (12/27)	0.137	0.041	33.7 (3/8)
3rd generation cephalosporin	11.0 (12/109)	11.8 (10/85)	0 (0/16)	0.357	38.9 (56/144)	46.2 (37/80)	25.9 (7/27)	0.063	<0.001	33.7 (3/8)
Penicillin + β lactamase inhibitor	14.7 (16/109)	12.9 (11/85)	31.2 (5/16)	0.066	15.3 (22/144)	12.5 (10/80)	14.8 (4/27)	0.758	0.985	12.5 (1/8)
Combination therapy	12.1 (15/124)	11.5 (11/96)	15.8 (3/19)		11.7 (19/163)	12.1 (11/91)	9.4 (5/32)			11.1 (1/9)
Change to 2nd regimen	8.9 (11/124)	8.3 (8/96)	10.5 (2/19)	0.757	14.7 (24/163)	18.7 (17/91)	9.4 (3/32)	0.220	0.133	33.3 (3/9)
Failure	2.4 (3/124)	2.1 (2/96)	5.3 (1/19)	0.421	2.5 (4/163)	1.1 (1/91)	- (0/32)	0.992	0.986	11.1 (1/9)
Death	2.6 (5/190)	1.9 (3/161)	9.1 (2/22)	0.051	3.5 (10/286)	3.8 (7/184)	5.7 (3/53)	0.554	0.597	0 (0/11)
Post-infectious complications (%)	0.6 (2/327) a	0.4 (1/284)	3.3 (1/30)	0.182	1.3 (5/379) b	0.8 (2/240)	0 (0/76)	0.634	0.460	0 (0/12)
Symptomatic relapse	4.2 (14/334)	3.1 (9/295)	22.6 (7/31)	<0.001	1.5 (6/389)	1.6 (4/246)	1.2 (1/80)	0.812	0.030	6.7 (1/15)
>1	28.6 (4/14)	33.3 (3/9)	14.3 (1/7)	0.383	16.7 (1/6)	0 (0/4)	100 (1/1)	0.346	0.573	0 (0/1)
Time from previous episode, mean (range), months	7.0 (0.5–23)	5.7 (0.5–19)	8.9 (1.5–23)	0.193	1.8 (0.5–6.5)	0.75 (0.5–1)	4.25 (2–6.5)	0.133	0.014	2.5
Immunosuppression	71.4 (10/14)	55.6 (5/9)	100 (7/7)	0.089	33.3 (2/6)	25.0 (1/4)	0 (0/1)	NA	0.161	100 (1/1)

Abbreviations: NA (not applicable), NTS (non-typhoidal Salmonella), TS (typhoidal Salmonella). ¹ Comparing Campylobacter jejuni and C. coli. ² Comparing Salmonella Typhimurium and S. enteritidis. ³ Comparing Campylobacter sp. and non-typhoidal Salmonella sp. ^a—1 Inflammatory bowel disease, 1 Guillain–Barré syndrome. ^b—2 inflammatory bowel disease, 3 reactive arthritis, 1 uveitis.

.

3.5. Reporting to the National Epidemiological Surveillance System

In total, 32.8% of cases of NTS infection, 30.6% of Campylobacter infections, and 14.3% of TS infections were reported to the SINAVE. The time from diagnosis to reporting ranged from 0 to 170 days and was significantly longer for Campylobacter infections than for NTS infections (median 9 vs. 6 days, p = 0.047) (

Table 5. Reporting to the National Epidemiological Surveillance System (SINAVE) of cases of Campylobacter and Salmonella infections diagnosed in a tertiary hospital in Northern Portugal, between 2015 and 2019, categorized by group (Campylobacter, non-typhoidal Salmonella, and typhoidal Salmonella), and species/serovar within each group.

	Campylobacter				NTS				p Value 3	TS
	All Species	C. jejuni	C. coli	p Value 1	All Serovars	S. typhimurium	S. enteritidis	p Value 2		All Serovars
All patients (n)	342	295	31		392	246	80			15
Reporting to SINAVE	30.6 (79/258)	31.2 (68/218)	36.0 (9/25)	0.625	32.8 (65/198)	34.7 (42/121)	32.6 (15/46)	0.798	0.615	14.3 (1/7)
Time after diagnosis, median (range), days	9 (3–170)	9 (3–170)	10 (4–43)	0.967	6 (2–62)	7 (2–62)	5 (2–57)	0.362	0.047	3 (0–7)

Abbreviations: NTS (non-typhoidal Salmonella), SINAVE (National Epidemiological Surveillance System), TS (typhoidal Salmonella). ¹ Comparing Campylobacter jejuni and C. coli. ² Comparing Salmonella Typhimurium and S. enteritidis. ³ Comparing Campylobacter sp. and non-typhoidal Salmonella sp.

). The percentage of cases reported by year to the SINAVE is represented in Figure 3c.

3.6. Associations Between Invasive Disease, Hospitalization, Reporting, and Sociodemographic and Clinical Factors

In the multivariate analysis (

Table 6. Potential factors associated with reporting of diagnosed cases of Salmonella sp. or Campylobacter sp. infection.

	Univariate			Multivariate
Potential Factors	Crude OR	95% CI	p Value	Adjusted OR	95% CI	p Value
Age ≥ 65 years old	1.24	0.78–1.97	0.369
Female sex	0.56	0.38–0.80	0.002 *	0.49	0.31–0.78	0.003 *
Immunosuppression	0.62	0.38–1.01	0.044 *	0.48	0.24–0.96	0.037 *
Hospitalisation	2.10	1.48–2.99	<0.001 *	2.08	1.34–3.24	0.001 *
Bacteremia/sepsis	1.18	0.68–2.02	0.559
Sample collected in ward	1.36	0.88–2.09	0.161
MDRO	0.53	0.33–0.86	0.010 *	0.46	0.28–0.76	0.003 *
Relapse	2.39	1.01–5.70	0.043 *	3.29	0.84–12.90	0.087
Death	0.99	0.31–3.13	0.988

Abbreviations: CI (confidence interval), MDRO (multidrug resistant organism), OR (odds ratio). * Statistically significant.

), reporting of cases diagnosed with Salmonella sp. or Campylobacter sp. infection was associated with male sex of the patient (OR 2.04, 95% CI 1.28–3.23, p = 0.003), no immunosuppression (OR 2.08, 95% CI 1.04–4.17, p = 0.037), hospitalization (OR 2.08, 95% CI 1.34–3.24, p = 0.001), and non-MDR organisms (OR 2.17, 95% CI 1.32–3.57, p = 0.003).

Invasive disease was only associated, in the multivariate analysis (

Table 7. Potential risk factors associated with invasive disease/hospitalization: (a) Campylobacter spp. infection; (b) Salmonella spp. infection.

Invasive Disease

Hospitalisation

(a)

Univariate

Multivariate

Univariate

Multivariate

Potential Factors

Crude OR

95% CI

p Value

Adjusted OR

95% CI

p Value

Crude OR

95% CI

p Value

Adjusted OR

95% CI

p Value

Age < 18 years old

0.83

0.76–0.90

<0.001 *

0.14

0.08–0.24

<0.001 *

Age ≥ 65 years old

3.13

1.01–10.10

0.045 *

1.00

0.26–3.95

0.995

8.31

3.74–18.47

<0.001 *

8.16

2.68–24.84

<0.001 *

Female sex

1.48

0.59–3.74

0.403

0.84

0.51–1.38

0.497

Immunosuppression

17.43

4.91–61.84

<0.001 *

17.42

4.83–62.82

<0.001*

2.93

1.74–4.93

<0.001 *

1.09

0.53–2.24

0.814

Bacteremia/sepsis

6.41

2.30–17.88

<0.001 *

8.18

2.16–30.93

0.002 *

Fever

0.82

0.32–2.13

0.681

1.39

0.84–2.30

0.204

AKI

0.94

0.73–1.58

0.216

10.74

3.83–30.11

<0.001 *

7.74

2.50–23.89

<0.001*

Invasive disease

Hospitalisation

(b)

Univariate

Multivariate

Univariate

Multivariate

Potential factors

Crude OR

95% CI

p value

Adjusted OR

95% CI

p value

Crude OR

95% CI

p value

Adjusted OR

95% CI

p value

Age < 18 years old

0.04

0.02–0.08

<0.001 *

0.31

0.20–0.48

<0.001 *

Age ≥ 65 years old

12.38

7.10–21.59

<0.001 *

0.93

0.8–11.25

0.957

3.64

2.11–6.29

<0.001 *

2.06

1.01–4.19

0.047 *

Female sex

1.60

0.99–2.57

0.054

0.79

0.10–6.08

0.818

0.60

0.40–0.89

0.011 *

0.63

0.39–1.01

0.055

Immunosuppression

3.98

2.17–7.28

<0.001 *

18.56

1.76–196.28

0.015 *

1.43

0.79–2.60

0.236

Bacteremia/sepsis

3.21

1.59–6.50

<0.001 *

1.09

0.42–2.84

0.865

Extraintestinal focal infection

1.12

0.60–2.11

0.717

Fever

0.44

0.26–0.72

0.001 *

0.21

0.02–2.13

0.187

1.47

0.95–2.28

0.085

2.01

1.14–3.54

0.016 *

AKI

4.64

2.63–8.16

<0.001 *

0.999

24.72

7.60–80.38

<0.001 *

19.85

5.85–67.38

<0.001 *

Fluoroquinole resistance

1.01

0.58–1.72

0.986

1.01

0.65–1.58

0.960

MDRO

0.64

0.38–1.08

0.090

0.65

0.05–8.62

0.747

0.96

0.64–1.45

0.849

Abbreviations: AKI (acute kidney injury), CI (confidence interval), MDRO (multidrug resistant organism), OR (odds ratio). * Statistically significant.

), with immunosuppression, both for Campylobacter infections (OR 17.42, 95% CI 4.83–62.83, p < 0.001) and for Salmonella infections (OR 18.56, 95% CI 1.76–196.28, p = 0.015). Factors associated with hospitalization of Campylobacter-infected patients in multivariate analysis were age over 64 years old (OR 8.16, 95% CI 2.68–24.84, p < 0.001), bacteremia/sepsis (OR 8.18, 95% CI 2.16–30.93, p = 0.002), and AKI (OR 7.74, 95% CI 2.50–23.89, p < 0.001). For Salmonella-infected patients, factors associated with hospitalization in multivariate analysis were also age over 64 years old (OR 2.06, 95% CI 1.01–4.19, p = 0.047) and AKI (OR 19.85, 95% CI 5.85–67.38, p < 0.001), as well as fever (OR 2.01, 95% CI 1.14–3.54, p = 0.016).

4. Discussion

This study analyzed 10 years of NTS infections and 5 years of Campylobacter infections in a tertiary hospital in Northern Portugal. Male sex and children were the groups predominantly affected by infection with both agents, as seen in previous national and European studies; a peak in cases of NTS between August and October was detected, but there was no clear seasonality for Campylobacter infections, which differs from European epidemiological data [32].

C. jejuni and C. coli were the main species of Campylobacter notified according to the EU One Health Zoonoses Report of 2021 [33], and the same trend was verified in the present study, with those two species accounting for 95.3% of the Campylobacter infections diagnosed. However, our data on Salmonella serovars differed from those reported in the EU One Health Zoonoses Report of 2021 [33], since the main serovar identified in our study was S. typhimurium (84.5% of cases), followed by S. enteritidis (8.9% of cases), versus a predominance of S. enteritidis (54.6%) in the EU. Our data, although different from the EU’s, are similar to other studies of NTS conducted in Portugal, which showed a predominance of monophasic variants of S. typhimurium 1,4,[5],12:i:- (26.6%), followed by Enteritidis (25.3%) and Typhimurium (14.7%) [34]. In this study, the identification of monophasic S. typhimurium was not feasible due to the unavailability of the requisite laboratory tests, so it was simply reported as S. typhimurium. Additionally, not all Salmonella specimens that were isolated underwent the typing process, which was inconclusive in other cases; therefore, some isolates were simply reported as Salmonella sp. (not excluding TS).

In 2020, the number of NTS infections was the lowest since 2010, which could be attributed to the COVID-19 pandemic. These findings are in accordance with the EU One Health Zoonoses Report of 2021, which also showed that the notification rate decreased by 19.6% and 23.1%, with and without the data for the United Kingdom, respectively [33]. Notably, there was an increasing trend in campylobacteriosis at our hospital. However, there are important laboratory conditions to take into account when interpreting these results. Certainly, the systematization of the use of an enteric transport medium (since 2016) and the modifications to the cultural exam protocol (incubation at 42 °C with a microaerophilic atmosphere) (since 2019) contributed to a greater recovery of Campylobacter sp.

The symptomatic presentation, in most patients, was associated with diarrhea, fever, nausea, and vomiting. However, more than 20% of NTS infections were invasive and up to 20% were associated with AKI, which was significantly associated with hospitalizations. Immunosuppression was the only factor in the multivariate analysis that was associated with invasive disease, which is similar to other studies in the EU [13,14]. It was also the main risk factor for relapse in Campylobacter infection. These data reinforce the need to consider Campylobacter and Salmonella infections in immunocompromised hosts, especially in cases of primary immunodeficiency for Campylobacter and secondary immunodeficiency for Salmonella (not only HIV infection but also inflammatory/autoimmune/neoplastic diseases that need immunosuppressive therapy). The most common form of extraintestinal focal disease was urinary infection, which can have implications in this group of patients, mainly in the differential diagnosis and choice of empiric antibiotic therapy (considering the high burden of resistance to fluoroquinolones).

Resistance to fluoroquinolones was high for both Campylobacter (96.2% of isolates tested), NTS (69.3%) and TS (78.6%), which is compatible with the resistances found in the EU in terms of campylobacteriosis—with an average level of ciprofloxacin resistance of 64.5% for C. jejuni and 69.6% for C. coli—but much higher than the EU average of 14.9% in Salmonella isolates—with the lowest levels observed in S. typhimurium (7.6%) and the S. typhimurium monophasic variant (8.9%)—and high to extremely high levels in S. infantis (33.9%) and S. kentucky (78.1%) [33]. These higher levels of resistance to quinolones could be related to a reported higher per capita consumption of these drugs in the community sector in Portugal, compared to most countries in Western and Northern Europe [35]. Antimicrobial susceptibility testing was performed in only half of the Campylobacter isolates. This is not ideal since it curtails surveillance and, in cases of invasive and recurrent infections, limits knowledge regarding the antibiotic susceptibility of the isolates, which, in turn, precludes appropriate antibiotic selection.

The multidrug resistance (MDR) phenotype was present in 37.8% of NTS isolates and was significantly more common in S. typhimurium (p < 0.001), which is also compatible with EU data, which demonstrated a trend towards high overall MDR (22.6%, n = 6867) among Salmonella spp., and MDR was most frequently reported among monophasic S. typhimurium 1,4,[5],12:i:- (78.4%, n = 871) [33]. The presence of MDR imposes limitations on available antimicrobial options; in this study, resistance to third-generation cephalosporins in Salmonella was low, so it seems to be the best option for invasive disease before antimicrobial susceptibility testing is available or if it is impossible to obtain. With regard to empiric treatment of acute diarrhea, according to our data, fluoroquinolones have very limited use in our population and macrolides appear to be a reasonable alternative.

There were fifteen cases of TS, only three (20%) of which had a history of travel abroad. This contrasts with reported data for the European region, where 92.4% of TS cases in 2019 were documented as related to travel abroad [36]. The ongoing detection of autochthonous cases, although rare, highlights the importance of serotyping every Salmonella isolate to increase early detection, ensure adequate treatment of TS, and allow directed public health interventions. The increasing population of migrants in Portugal, including from highly TS endemic countries in South Asia [37], could represent a direct or environmental source for TS. Systematic screening programs for migrants could help reduce the risk of autochthonous transmission and increase the overall health conditions of migrants.

The 2017 EFSA report [32] showed a notification rate of Salmonella infection in Portugal of 4.5 confirmed cases/100,000, which was inferior to the average in the EU (19.7/100,000). Considering the high prevalence of NTS and Campylobacter in food and animal products and the steep discrepancy between the incidence rates of these infections in Portugal compared with other EU countries, there is a high probability of underreporting of cases. Indeed, our study showed that less than one third of the NTS and Campylobacter infections diagnosed were reported to SINAVE. These findings are also in accordance with another study conducted in Portugal by Morgado et al. [30] in 2015, who developed a model that generated occurrences of salmonellosis cases 93 times higher than those reported by the surveillance system (239 cases of salmonellosis reported vs. 22,201 cases estimated in 2010). The underreporting probably reflects a lack of time, unawareness about mandatory notification, and the lengthy process that is involved in notification. The information collected in the present study could be used to improve reporting by investing in simpler ways of notifying cases and in increasing awareness among healthcare professionals.

The global impact on morbidity and the economic losses of salmonellosis and campylobacteriosis are significant. Even so, the epidemiological and clinical aspects of these infections remain largely unknown, likely because many patients do not seek medical attention and, even when they do so, an empirical treatment approach to gastroenteritis is followed, without sample collection or microbiological identification. Additionally, many cases are recognized but not reported as part of an integrated surveillance network. The improvement in diagnosis and reporting could provide a wider picture of campylobacteriosis and salmonellosis and support the establishment and improvement of food safety programs, especially since these infectious agents are zoonotic.

An important limitation of this study is that it was a retrospective study and some of the data requested were not available in clinical records. Another limitation was the fact that the techniques for identification of Campylobacter changed over time, which can explain the increase in incidence from 2019 onwards. The data and conclusions from this study cannot be generalized to the Portuguese population, since the data were only collected from a specific region of the country and from patients that presented themselves to the hospital, which will likely cause a relative increase of diagnoses in children and reflect only severe cases in adults.

5. Conclusions

This study shows that immunosuppressive conditions are common in hospital diagnosed cases and are associated with relapsing and invasive disease. Unawareness of invasive presentations can make diagnosis and appropriate treatment challenging. Acute kidney injury is a common complication of infection and is associated with the need for hospital admission. High rates of multidrug resistance limit therapeutic options. Underreporting is substantial and should be addressed to allow a more comprehensive picture of disease at a national level. It is expected that these results will bring forward the need for change in reporting practices and that they will provide a stepping stone for a national-level study to better comprehend the trends of these diseases and their impact on the Portuguese population.